Protein kinase C inhibitors and uses thereof

ABSTRACT

This disclosure concerns compounds which are useful as inhibitors of protein kinase C (PKC) and are thus useful for treating a variety of diseases and disorders that are mediated or sustained through the activity of PKC. This disclosure also relates to pharmaceutical compositions comprising these compounds, methods of using these compounds in the treatment of various diseases and disorders, processes for preparing these compounds and intermediates useful in these processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/687,054 filed on Jan. 13, 2010, which claims priority benefit under35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/145,021,filed on Jan. 15, 2009 and U.S. Provisional Patent Application No.61/147,353, filed on Jan. 26, 2009, which are incorporated by referencein their entireties.

BACKGROUND

Protein kinase C (“PKC”) is a key enzyme in signal transduction involvedin a variety of cellular functions, including cell growth, regulation ofgene expression, and ion channel activity. The PKC family of isozymesincludes at least 11 different protein kinases that can be divided intoat least three subfamilies based on their homology and sensitivity toactivators. Each isozyme includes a number of homologous (“conserved” or“C”) domains interspersed with isozyme-unique (“variable” or “V”)domains. Members of the “classical” or “cPKC” subfamily, PKC α, β_(i),β_(ii) and γ, contain four homologous domains (C1, C2, C3 and C4) andrequire calcium, phosphatidylserine, and diacylglycerol or phorbolesters for activation. Members of the “novel” or “nPKC” subfamily, PKCβ, ε, η and θ, lack the C2 homologous domain and do not require calciumfor activation. Finally, members of the “atypical” or “αPKC” subfamily,PKC ζ and λ/i, lack both the C2 and one-half of the C1 homologousdomains and are insensitive to diacylglycerol, phorbol esters andcalcium.

SUMMARY

This disclosure concerns compounds which are useful as inhibitors ofprotein kinase C(PKC) and are thus useful for treating a variety ofdiseases and disorders that are mediated or sustained through theactivity of PKC. This disclosure also relates to pharmaceuticalcompositions comprising these compounds, methods of using thesecompounds in the treatment of various diseases and disorders, processesfor preparing these compounds and intermediates useful in theseprocesses.

Exemplary chemical structures are provided throughout the disclosure. Byway of example, such compounds are represented by the following formula(I):

wherein

R¹ is selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,—C(O)OR^(1a), —S(O)R^(1b), and —S(O)₂R^(1c); wherein each of R^(1a),R^(1b), and R^(1c) is independently hydrogen, alkyl or phenyl-alkyl;

R^(a), R^(b), R^(c) and R^(d) independently are selected from hydrogenand alkyl;

m is an integer from one to five;

p is an integer from zero to six;

R² is selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy,substituted alkyl, substituted alkoxy, amino, substituted amino,aminoacyl, acylamino, azido, carboxyl, carboxylalkyl, cyano, halogen,nitro, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl, and trihalomethyl;

X¹, X², and X³ are CR⁵ or one of X¹, X², and X³ is N and rest are CR⁵;

R⁵ is selected from hydrogen, halogen, alkyl and substituted alkyl;

R³ and R⁴ are, for each occurrence, independently selected fromhydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl; or R³ and R⁴ together with the carbon atom to whichthey are attached form a carbocyclic or heterocyclic 4 to 8-memberedring;

n is an integer from one to three;

Z¹, Z², and Z³ are selected from CR⁶R^(6a), N, O, and S;

Z⁴ and Z⁵ are selected from N, C, and CR⁶;

R⁶ is selected from hydrogen, halogen, alkyl and substituted alkyl;

R^(6a) is selected from hydrogen, halogen, alkyl and substituted alkylor is absent to satisfy valence requirements; and

the dashed lines represent a single bond or double bond;

or a salt or solvate or stereoisomer thereof.

DEFINITIONS

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain, e.g., having from 1 to 40 carbon atoms,from 1 to 10 carbon atoms, or from 1 to 6 carbon atoms. This term isexemplified by groups such as methyl, ethyl, n-propyl, iso-propyl,n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl, and the like.

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

The term “alkylene” refers to a diradical of a branched or unbranchedsaturated hydrocarbon chain, usually having from 1 to 40 carbon atoms,more usually 1 to 10 carbon atoms and even more usually 1 to 6 carbonatoms. This term is-exemplified by groups such as methylene (—CH₂—),ethylene (—CH₂CH₂—), the propylene isomers (e.g., —CH₂CH₂CH₂— and—CH(CH₃)CH₂—) and the like.

The term “substituted alkylene” refers to an alkylene group as definedherein wherein one or more carbon atoms in the alkylene chain have beenoptionally replaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)—(where n is 0 to 2), —NR— (where R is hydrogen or alkyl) and having from1 to 5 substituents selected from the group consisting of alkoxy,substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl,aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo,thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-aryl, —SO₂-heteroaryl, and —NR′R″, wherein R′ and R″may be the same or different and are chosen from hydrogen, optionallysubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl,heteroaryl and heterocyclic.

The term “alkane” refers to alkyl group and alkylene group, as definedherein.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl”refers to the groups R′NHR″— where R′ is alkyl group as defined hereinand R″ is alkylene, alkenylene or alkynylene group as defined herein.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

The term “alkoxy” refers to the groups alkyl-O—, alkenyl-O—,cycloalkyl-O—, cycloalkenyl-O—, and alkynyl-O—, where alkyl, alkenyl,cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkoxyamino” refers to the group —NH-alkoxy, wherein alkoxy isdefined herein.

The term “haloalkoxy” refers to the groups alkyl-O— wherein one or morehydrogen atoms on the alkyl group have been substituted with a halogroup and include, by way of examples, groups such as trifluoromethoxy,and the like.

The term “haloalkyl” refers to a substituted alkyl group as describedabove, wherein one or more hydrogen atoms on the alkyl group have beensubstituted with a halo group. Examples of such groups include, withoutlimitation, fluoralkyl groups, such as trifluoromethyl, difluoromethyl,trifluoroethyl and the like.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkenyl” refers to a monoradical of a branched or unbranchedunsaturated hydrocarbon group having from 2 to 40 carbon atoms, from 2to 10 carbon atoms, or from 2 to 6 carbon atoms and having at least 1site (e.g., from 1-6 sites) of vinyl unsaturation.

The term “substituted alkenyl” refers to an alkenyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbonhaving from 2 to 40 carbon atoms, from 2 to 20 carbon atoms, or from 2to 6 carbon atoms and having at least 1 site (e.g., from 1-6 sites) ofacetylene (triple bond) unsaturation.

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

The term “acyl” refers to the groups HC(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—,cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—, aryl-C(O)—,heteroaryl-C(O)— and heterocyclic-C(O)— where alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.

The term “acylamino” or “aminocarbonyl” refers to the group —C(O)NRRwhere each R is independently hydrogen, alkyl, substituted alkyl, aryl,heteroaryl, heterocyclic or where both R groups are joined to form aheterocyclic group (e.g., morpholino) wherein alkyl, substituted alkyl,aryl, heteroaryl, and heterocyclic are as defined herein.

The term “aminoacyl” refers to the group —NRC(O)R where each R isindependently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, orheterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl, andheterocyclic are as defined herein.

The term “aminoacyloxy” or “alkoxycarbonylamino” refers to the group—NRC(O)OR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclic-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclic are as defined herein.

The term “aryl” refers to an unsaturated aromatic carbocyclic group offrom 6 to 20 carbon atoms having a single ring (e.g., phenyl) ormultiple condensed (fused) rings (e.g., naphthyl or anthryl). Exemplaryaryls include phenyl, naphthyl and the like. Unless otherwiseconstrained by the definition for the aryl substituent, such aryl groupscan optionally be substituted with from 1 to 5 substituents, or from 1to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.

The term “aryloxy” refers to the group aryl-O— wherein the aryl group isas defined herein including optionally substituted aryl groups as alsodefined herein.

The term “amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclic provided that atleast one R is not hydrogen.

The term “azido” refers to the group —N₃.

The term “carboxyl” refers to —COOH or salts thereof.

The term “carboxyalkyl” or “carboxylalkyl” or “alkoxycarbonyl” refers tothe groups “—C(O)O-alkyl”, “—C(O)O-substituted alkyl”,“—C(O)β-cycloalkyl”, “—C(O)O-substituted cycloalkyl”, “—C(O)O-alkenyl”,“—C(O)O-substituted alkenyl”, “—C(O)O-alkynyl” and “—C(O)O— substitutedalkynyl” where alkyl, substituted alkyl, cycloalkyl, substitutedcycloalkyl, alkenyl, substituted alkenyl, alkynyl and substitutedalkynyl alkynyl are as defined herein.

The term “cyano” refers to the group —CN.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 20carbon atoms having a single cyclic ring and at least one point ofinternal unsaturation. Examples of suitable cycloalkenyl groups include,for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl, andthe like.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbonatoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfurwithin at least one ring (if there is more than one ring). Unlessotherwise constrained by the definition for the heteroaryl substituent,such heteroaryl groups can be optionally substituted with 1 to 5substituents, or from 1 to 3 substituents, selected from acyloxy,hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, substituted alkyl, substituted alkoxy, substitutedalkenyl, substituted alkynyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro,heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy,oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl, and trihalomethyl.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl wherealkylene and heteroaryl are defined herein. Such heteroaralkyl groupsare exemplified by pyridylmethyl, pyridylethyl, indolylmethyl, and thelike.

The term “heteroaryloxy” refers to the group heteroaryl-O—.

The term “heterocycle” or “heterocyclic” refers to a monoradicalsaturated or unsaturated group having a single ring or multiplecondensed rings, from 1 to 40 carbon atoms and from 1 to 10 heteroatoms, e.g., from 1 to 4 heteroatoms, selected from nitrogen, sulfur,phosphorus, and/or oxygen within the ring. Unless otherwise constrainedby the definition for the heterocyclic substituent, such heterocyclicgroups can be optionally substituted with 1 to 5, or from 1 to 3substituents, selected from alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl, andfused heterocycle.

Examples of nitrogen heteroaryls and heterocycles include, but are notlimited to, pyrrole, thiophene, furan, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, pyrrolidine, piperidine, piperazine,indoline, morpholine, tetrahydrofuranyl, tetrahydrothiophene, and thelike as well as N-alkoxy-nitrogen containing heterocycles.

The term “heterocyclooxy” refers to the group heterocyclic-O—.

The term “heterocyclothio” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein, and is exemplified by the groups2,6-morpholino, 2,5-morpholino and the like.

The term “hydroxyamino” refers to the group —NHOH.

The term “oxo” refers to the group ═O.

The term “oxyacylamino” or “aminocarbonyloxy” refers to the group—OC(O)NRR where each R is independently hydrogen, alkyl, substitutedalkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substitutedalkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term “thiol” refers to the group —SH.

The term “thioalkoxy” or “alkylthio” refers to the group —S-alkyl.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined herein including optionally substituted aryl groupsalso defined herein.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined herein including optionallysubstituted aryl groups as also defined herein.

The term “thioheterocyclooxy” refers to the group heterocyclyl-S—wherein the heterocyclyl group is as defined herein including optionallysubstituted heterocyclyl groups as also defined herein.

The term “thioketo” refers to the group ═S.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (e.g.,salts having acceptable mammalian safety for a given dosage regime).Such salts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation, such as a metal cation or an organiccation and the like. Where applicable, the salt is a pharmaceuticallyacceptable salt, although this is not required for salts of intermediatecompounds that are not intended for administration to a patient.

The term “solvate” as used herein refers to a complex or aggregateformed by one or more molecules of a solute, i.e. a compound of theinvention or a pharmaceutically-acceptable salt thereof, and one or moremolecules of a solvent. Such solvates are typically crystalline solidshaving a substantially fixed molar ratio of solute and solvent.Representative solvents include by way of example, water, methanol,ethanol, isopropanol, acetic acid, and the like. When the solvent iswater, the solvate formed is a hydrate.

It will be appreciated that the term “or a salt or solvate orstereoisomer thereof” is intended to include all permutations of salts,solvates and stereoisomers, such as a solvate of a pharmaceuticallyacceptable salt of a stereoisomer of a compound of formula (I).

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition in a patient, such as amammal (particularly a human) that includes: (a) preventing the diseaseor medical condition from occurring, i.e., prophylactic treatment of apatient; (b) ameliorating the disease or medical condition, i.e.,eliminating or causing regression of the disease or medical condition ina patient; (c) suppressing the disease or medical condition, i.e.,slowing or arresting the development of the disease or medical conditionin a patient; or (d) alleviating the symptoms of the disease or medicalcondition in a patient.

DETAILED DESCRIPTION

This disclosure concerns compounds which are useful as inhibitors ofprotein kinase C(PKC) and are thus useful for treating a variety ofdiseases and disorders that are mediated or sustained through theactivity of PKC. This disclosure also relates to pharmaceuticalcompositions comprising these compounds, methods of using thesecompounds in the treatment of various diseases and disorders, processesfor preparing these compounds and intermediates useful in theseprocesses.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbookof Practical Organic Chemistry, Including Qualitative Organic Analysis,Fourth Edition, New York: Longman, 1978.

The nomenclature used herein to name the subject compounds isillustrated in the Examples herein. This nomenclature has generally beenderived using the commercially-available AutoNom software (MDL, SanLeandro, Calif.).

REPRESENTATIVE EMBODIMENTS

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments. Theserepresentative values are intended to further define and illustrate suchaspects and embodiments and are not intended to exclude otherembodiments or to limit the scope of this invention. In this regard, therepresentation that a particular value or substituent is preferred isnot intended in any way to exclude other values or substituents fromthis invention unless specifically indicated.

These compounds may contain one or more chiral centers and therefore,the embodiments are directed to racemic mixtures; pure stereoisomers(i.e., enantiomers or diastereomers); stereoisomer-enriched mixtures andthe like unless otherwise indicated. When a particular stereoisomer isshown or named herein, it will be understood by those skilled in the artthat minor amounts of other stereoisomers may be present in thecompositions unless otherwise indicated, provided that the desiredutility of the composition as a whole is not eliminated by the presenceof such other isomers.

In one of its composition aspects, the present embodiments provide acompound of formula (I):

wherein

R¹ is selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,—C(O)OR^(1a), —S(O)R^(1b), and —S(O)₂R^(1c); wherein each of R^(1a),R^(1b), and R^(1c) is independently hydrogen, alkyl or phenyl-alkyl;

R^(a), R^(b), R^(c) and R^(d) independently are selected from hydrogenand alkyl;

m is an integer from one to five;

p is an integer from zero to six;

R² is selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy,substituted alkyl, substituted alkoxy, amino, substituted amino,aminoacyl, acylamino, azido, carboxyl, carboxylalkyl, cyano, halogen,nitro, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl, and trihalomethyl;

X¹, X², and X³ are CR⁵ or one of X¹, X², and X³ is N and rest are CR⁵;

R⁵ is, for each occurrence, selected from hydrogen, halogen, alkyl andsubstituted alkyl;

R³ and R⁴ are, for each occurrence, independently selected fromhydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl; or R³ and R⁴ together with the carbon atom to whichthey are attached form a carbocyclic or heterocyclic 4 to 8-memberedring;

n is an integer from zero to three;

Z¹, Z², and Z³ are selected from CR⁶R^(6a), N, O, and S;

Z⁴ and Z⁵ are selected from N, C, and CR⁶;

R⁶ is selected from hydrogen, halogen, alkyl and substituted alkyl;

R^(6a) is selected from hydrogen, halogen, alkyl and substituted alkylor is absent to satisfy valence requirements; and

the dashed lines represent a single bond or double bond;

or a salt or solvate or stereoisomer thereof.

The groups Z¹, Z², Z³, Z⁴, and Z⁵ and the sites of the optional doublebonds indicated by the dashed lines in the formulas are selected suchthat the ring containing these groups satisfies valence requirements.For example, the ring may contain one or two double bonds or no boublebonds as indicated by the dashed lines in this ring. If the ring isaromatic, the double bonds are positioned properly and the number ofsubstituents on Z¹, Z², Z³, Z⁴, and Z⁵ are such that aromaticity occurs.

R¹ can be selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,—C(O)OR^(1a), —S(O)R^(1b), and —S(O)₂R^(1c); wherein each of R^(1a),R^(1b), and R^(1c) is independently hydrogen, alkyl or phenyl-alkyl. Incertain instances, R¹ is hydrogen, alkyl, or cycloalkyl. In certaininstances, R¹ is alkenyl or alkynyl. In certain instances, R¹ is—C(O)OR^(1a), —S(O)R^(1b), or —S(O)₂R^(1c); wherein each of R^(1a),R^(1b), and R^(1c) is independently hydrogen, alkyl or phenyl-alkyl. Incertain cases, R¹ is selected from hydrogen and alkyl. In one case, R¹is hydrogen. In one case, R¹ is alkyl. In one case, R¹ is methyl.

R^(a), R^(b), R^(c), and R^(d) independently are selected from hydrogenand alkyl. In certain cases, at least one or two of R^(a), R^(b), R^(c),and R^(d) are lower alkyl. In certain instances, R^(a), R^(b), R^(c),and R^(d) represent lower alkyl groups. In certain instances, R^(a),R^(b), R^(c), and R^(d) represent methyl. In certain instances, R^(a),R^(b), R^(c), and R^(d) represent hydrogen.

The value for m can be from one to five. In certain instances, m is aninteger from one to three. In one case, m is one or two. In one case, mis one. In one case, m is two.

The value for p can be an integer from zero to six. In certaininstances, p is an integer from zero to four or an integer from zero tothree. In one case, p is zero or one. In one case, p is zero. In onecase, p is one.

R² can be selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy,substituted alkyl, substituted alkoxy, amino, substituted amino,aminoacyl, acylamino, azido, carboxyl, carboxylalkyl, cyano, halogen,nitro, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl, and trihalomethyl.In certain instances, R² is hydroxy, alkyl, substituted alkyl, alkoxy,substituted alkoxy, cyano, halogen, nitro, trihalomethyl, acyloxy, acyl,or acylamino. In certain instances, R² is thiol, amino, substitutedamino, aminoacyl, aminoacyloxy, oxyacylamino, or thioalkoxy, substitutedthioalkoxy. In certain instances, R² is azido, carboxyl, carboxylalkyl,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, or —SO₂-heteroaryl. In one case, R²is hydroxy, alkyl, alkoxy, cyano, halogen, nitro, or trihalomethyl. Inone case, R² is halogen. In one case, R² is fluoro. In one case, R² ishydroxyl. R² is alkyl.

With continued reference to formula (I), X¹, X², and X³ can be CR⁵ orone of X¹, X², and X³ can be N and rest are CR⁵; where R⁵ for eachoccurrence independently is selected from hydrogen, halogen, alkyl andsubstituted alkyl. In certain instances, X¹, X², and X³ is CR⁵, where R⁵for each occurrence is selected from hydrogen, halogen, alkyl andsubstituted alkyl. In some cases, X¹, X², and X³ is CR⁵, where R⁵ foreach occurrence is hydrogen, alkyl, or substituted alkyl. In some cases,X¹, X², and X³ is CR⁵, where R⁵ for each occurrence is hydrogen, fluoro,alkyl or haloalkyl. In certain instances, X¹, X², and X³ are CH. Incertain instances, one of X¹, X², and X³ is N and rest are CR⁵. In onecase, X¹ is N. In one case, X² is N. In one case, X³ is N.

The value for n can be from one to three. In one case, n is one or two.In one case, n is one. In one case, n is two.

R³ and R⁴ can be, for each occurrence, independently selected fromhydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl; or R³ and R⁴ together with the carbon atom to whichthey are attached form a carbocyclic or heterocyclic 4 to 8-memberedring.

In certain instances, R³ and R⁴ can be, for each occurrence,independently selected from hydrogen, alkyl, substituted alkyl, alkoxy,substituted alkoxy, acyl, acylamino, acyloxy, cyano, halogen, hydroxyl,carboxyl, carboxylalkyl, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. In certain cases,R³ and R⁴ are, for each occurrence, independently selected fromhydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cyano,halogen, hydroxyl, and nitro. In certain cases, R³ and R⁴ are, for eachoccurrence, independently selected from hydrogen, alkyl, and halogen.

In certain instances, R³ and R⁴ together with the carbon atom to whichthey are attached form a carbocyclic or heterocyclic 4 to 8-memberedring. In certain cases, R³ and R⁴ together with the carbon atom to whichthey are attached form a carbocyclic or heterocyclic 4 or 5-memberedring. In certain instances, R³ and R⁴ together with the carbon atom towhich they are attached form a carbocyclic or heterocyclic 6-memberedring. In certain instances, R³ and R⁴ together with the carbon atom towhich they are attached form a carbocyclic or heterocyclic 7 or8-membered ring. In certain cases, the ring is carbocyclic. In certaincases, the ring is heterocyclic, such as a ring comprising O, S or N.

Z¹, Z², and Z³ can be selected from CR⁶, N, O, and S; where R⁶ isselected from hydrogen, halogen, alkyl and substituted alkyl; and thedashed lines represent a single bond or double bond. In certaininstances, Z¹, Z², and Z³ can be selected from CR⁶ and N. In certaininstances, Z¹, Z², and Z³ can be selected from CH and N. In certaininstances, Z¹, Z², and Z³ can be selected from CR⁶ and O. In certaininstances, Z¹, Z², and Z³ can be selected from CR⁶ and S. In certaincases, Z¹, Z², and Z³ are each N. In certain cases, Z¹, Z², and Z³ areeach CH. When Z¹, Z², or Z³ is S or N, these atoms can optionally besubstituted with one or more oxygen atoms. For example Z¹, Z², or Z³independently can be a sulfoxide [S(O) or sulfonyl S(O)₂] group.Similarly, Z¹, Z², and Z³ also can represent an N-oxide group, (N→O).

Z⁴ and Z⁵ are selected from N, C, and CR⁶. In certain instances, Z⁴ isN. In certain instances, Z⁴ is C. In certain instances, Z⁴ is CR⁶. Incertain instances, Z⁵ is N. In certain instances, Z⁵ is C. In certaininstances, Z⁵ is CR⁶.

A group of compounds of interest are compounds of formula (I), whereinX¹, X², and X³ are CH; and R² is fluoro. In certain cases, in formula(I), X¹, X², and X³ are CH; R² is fluoro; and R³ and R⁴ are hydrogen,methyl, or fluoro. In certain cases, in formula (I), X¹, X², and X³ areCH; R² is fluoro; and Z¹, Z², and Z³ are each N. In certain cases, informula (I), X¹, X², and X³ are CH; R² is fluoro; and Z¹, Z², and Z³ areeach CH.

Another group of compounds of interest are compounds of formula (I),wherein m is 2; and p is zero. In certain cases, in formula (I), m is 2;p is zero; and R² is fluoro.

Another group of compounds of interest are compounds of formula (I),wherein m is one; and p is one. In certain cases, in formula (I), m isone; p is one; and R² is fluoro.

Another group of compounds of interest are compounds of formula (I),wherein X² is N; X¹ and X³ are CH; and R² is fluoro. In certain cases,in formula (I), X² is N; X¹ and X³ are CH; R² is fluoro; and R³ and R⁴are hydrogen, methyl, or fluoro. In certain cases, in formula (I), X² isN; X¹ and X³ are CH; R² is fluoro; and Z¹, Z², and Z³ are each N. Incertain cases, in formula (I), X² is N; X¹ and X³ are CH; R² is fluoro;and Z¹, Z², and Z³ are each CH.

Another group of compounds of interest are compounds of formula (I),wherein X³ is N; X¹ and X² are CH; and R² is fluoro. In certain cases,in formula (I), X³ is N; X¹ and X² are CH; R² is fluoro; and R³ and R⁴are hydrogen, methyl, or fluoro. In certain cases, in formula (I), X³ isN; X¹ and X² are CH; R² is fluoro; and Z¹, Z², and Z³ are each N. Incertain cases, in formula (I), X³ is N; X¹ and X² are CH; R² is fluoro;and Z¹, Z², and Z³ are each CH.

Another group of compounds of interest are compounds of formula (I),wherein n is one; and R³ and R⁴ are, for each occurrence, independentlyselected from hydrogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, cyano, halogen, hydroxyl, and nitro.

Another group of compounds of interest are compounds of formula (I),wherein n is one; and R³ and R⁴ are, for each occurrence, independentlyselected from hydrogen, alkyl, and halogen.

Another group of compounds of interest are compounds of formula (I),wherein n is one; and R³ and R⁴ are the same and are selected fromhydrogen, alkyl, and halogen.

Another group of compounds of interest are compounds of formula (I),wherein n is 1; and one of R³ and R⁴ is hydrogen and the other isselected from alkyl and halogen. In certain cases, the compound isenantiomerically enriched (R)-configuration or (S)-configuration at thecarbon comprising R³ and R⁴. In certain cases, the compound is racemic.

Another group of compounds of interest are compounds of formula (I),wherein n is 2; and R³ and R⁴ are, for each occurrence, independentlyselected from hydrogen, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, cyano, halogen, hydroxyl, and nitro.

Another group of compounds of interest are compounds of formula (I),wherein n is 2; and R³ and R⁴ are, for each occurrence, independentlyselected from hydrogen, alkyl, and halogen.

Another group of compounds of interest are compounds of formula (I),wherein n is 2; and R³ and R⁴ on the same carbon are the same and areselected from hydrogen, alkyl, and halogen.

Another group of compounds of interest are compounds of formula (I),wherein n is 2; and on a given carbon comprising R³ and R⁴, one of R³and R⁴ is hydrogen and the other is selected from alkyl and halogen. Incertain cases, the compound is enantiomerically enriched. In certaincases, the compound is racemic.

Certain compounds of interest have formula (I) wherein R^(a), R^(b),R^(c) and R^(d) represent lower alkyl groups. Particular examples ofsuch compounds include those wherein R^(a), R^(b), R^(c) and R^(d) aremethyl groups and have formula (II):

A particular group of compounds of interest are compounds of formula(I), wherein X¹, X², and X³ are each CH. These compounds have thefollowing formula (III):

A particular group of compounds of interest are compounds of formula(I), wherein X¹, X², and X³ are each CH; and m is 2. These compoundshave the following formula (IV):

A particular group of compounds of interest are compounds of formula(I), wherein X¹, X², and X³ are each CH; and m is one. These compoundshave the following formula (V):

A particular group of compounds of interest are compounds of formula(I), wherein X¹, X², and X³ are each CH; n is 2; and one set of R³ andR⁴ is hydrogen. These compounds have the following formula (VI):

A particular group of compounds of interest are compounds of formula(I), wherein X² is N and X¹ and X³ are each CH. These compounds have thefollowing formula (VII):

A particular group of compounds of interest are compounds of formula(I), wherein X³ is N and X¹ and X² are each CH. These compounds have thefollowing formula (VIII):

An additional group of compounds of interest have formula (I), whereinZ⁴ is C and Z⁵ is N. Such compounds have the following formula (IX):

Particular compounds of interest are shown in the following tables.

TABLE 1

Compound R¹ R² R³ R⁴ Ring 1 Ring 2 1-1  —H   —F —H   —H  

1-2  —CH₃ —F —H   —H  

1-3  —H   —F —H   —H  

1-4  —CH₃ —F —H   —H  

1-5  —H   —F —F   —F  

1-6  —CH₃ —F —F   —F  

1-7  —H   —F —CH₃ —CH₃

1-8  —CH₃ —F —CH₃ —CH₃

1-9  —H   —F —CH₃ —CH₃

1-10 —CH₃ —F —CH₃ —CH₃

1-11 —H   —F

1-12 —CH₃ —F

1-13 —H   —F

1-14 —CH₃ —F

1-15 —H   —F —CH₃ —H  

1-16 —CH₃ —F —CH₃ —H  

1-17 —H   —F (R,S)-CH₃ —H  

1-18 —CH₃ —F (R,S)-CH₃ —H  

1-19 —H   —F (R)-CH₃ —H  

1-20 —CH₃ —F (R)-CH₃ —H  

1-21 —H   —F (S)-CH₃ —H  

1-22 —CH₃ —F (S)-CH₃ —H  

1-23 —H   —F —CH₃ —CH₃

1-24 —CH₃ —F —CH₃ —CH₃

1-25 —H   —F —CH₃ —CH₃

1-26 —CH₃ —F —CH₃ —CH₃

TABLE 2

Compound R¹ R² R³ R⁴ Ring 1 Ring 2 * center 2-1  —H   —F —H   —H  

2-2  —CH₃ —F —H   —H  

2-3  —H   —F —H   —H  

2-4  —CH₃ —F —H   —H  

2-5  —H   —F —F   —F  

2-6  —CH₃ —F —F   —F  

2-7  —H   —F —CH₃ —CH₃

* center is (S) (+) 2-8  —H   —F —CH₃ —CH₃

* center is (R) (−) 2-9  —CH₃ —F —CH₃ —CH₃

* center is (S) (+) 2-10 —CH₃ —F —CH₃ —CH₃

* center is (R) (−) 2-11 —H   —F —CH₃ —CH₃

2-12 —CH₃ —F —CH₃ —CH₃

2-13 —H   —F —CH₃ —CH₃

2-14 —CH₃ —F —CH₃ —CH₃

2-15 —H   —F

2-16 —CH₃ —F

2-17 —H   —F

2-18 —CH₃ —F

2-19 —H   —F —CH₃ —H  

2-20 —CH₃ —F —CH₃ —H  

2-21 —H   —F (R,S)-CH₃ —H  

2-22 —CH₃ —F (R,S)-CH₃ —H  

2-23 —H   —F (R)-CH₃ —H  

2-24 —CH₃ —F (R)-CH₃ —H  

2-25 —H   —F (S)-CH₃ —H  

2-26 —CH₃ —F (S)-CH₃ —H  

2-27 —H   —F —CH₃ —CH₃

2-28 —CH₃ —F —CH₃ —CH₃

2-29 —H   —F —CH₃ —CH₃

2-30 —CH₃ —F —CH₃ —CH₃

TABLE 3

Compound R¹ R² R³ R⁴ Ring 1 Ring 2 3-1 —H   —F —CH₃ —CH₃

3-2 —CH₃ —F —CH₃ —CH₃

Particular compounds of interest, and salts or solvates or stereoisomersthereof, include:

-   N2-(4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(4H-benzo[b]pyrrolo[1,2-d][1,4]oxazin-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(4H-benzo[b]pyrrolo[1,2-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(4,4-difluoro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(4,4-difluoro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(5,5-dimethyl-5H-benzo[e]tetrazolo[1,5-c][1,3]oxazin-9-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(5,5-dimethyl-5H-benzo[e]tetrazolo[1,5-c][1,3]oxazin-9-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(8,9-dihydrospiro[benzo[b]tetrazolo[1,5-d][1,4]oxazine-4,1′-cyclobutane]-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(8,9-dihydrospiro[benzo[b]tetrazolo[1,5-d][1,4]oxazine-4,1′-cyclobutane]-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   5-fluoro-N2-(4-methyl-8,9-dihydro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   5-fluoro-N2-(4-methyl-8,9-dihydro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine;-   N2-(4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-((1,2,2,5,5-pentamethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine;-   N2-(4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-((2,2,5,5-tetramethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine;-   N2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-((1,2,2,5,5-pentamethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine;-   N2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-((2,2,5,5-tetramethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine;-   N2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(((3S)-2,2,5-trimethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine;    and-   N2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(((3R)-2,2,5-trimethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine.

The present disclosure also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof.

A disclosed compound can be administered alone, as the sole activepharmaceutical agent, or in combination with one or more additionalcompounds of formula (I) or in conjunction with other agents. Whenadministered as a combination, the therapeutic agents can be formulatedas separate compositions that are administered simultaneously or atdifferent times, or the therapeutic agents can be administered togetheras a single composition combining two or more therapeutic agents. Thus,the pharmaceutical compositions disclosed herein containing a compoundof formula (I) optionally contain other therapeutic agents. Accordingly,certain embodiments are directed to such pharmaceutical composition,wherein the composition further comprises a therapeutically effectiveamount of an agent selected as is known to those of skill in the art.

The subject compounds can inhibit a protein kinase C activity.Accordingly, the compounds are useful for treating a disease or disorderthat is mediated through the activity of a PKC activity in a subject.Also, the compounds are useful for treating a disease or disorder thatis associated with the activation of T-cells in a subject.

The present disclosure provides a method of treating an inflammatorydisease in a subject, the method comprising administering to the subjectwith a compound of formula (I) or a salt or solvate or stereoisomerthereof.

The present disclosure also provides a method of treating an autoimmunedisease in a subject, the method comprising administering to the subjectwith a compound of formula (I) or a salt or solvate or stereoisomerthereof.

The present disclosure also provides a method of treating an oculardisease or disorder involving inflammatory and/or neovascular events.

The present disclosure also provides a method of treating diseases orconditions of interest including, but are not limited to,atherosclerosis, vascular occlusion due to vascular injury, angioplasty,restenosis, obesity, syndrome X, impaired glucose tolerance, polycysticovary syndrome, hypertension, heart failure, chronic obstructivepulmonary disease, CNS diseases, Alzheimer disease, amyotrophic lateralsclerosis, cancer, infectious disease, AIDS, septic shock, adultrespiratory distress syndrome, ischemia/reperfusion injury, myocardialinfarction, stroke, gut ischemia, renal failure, hemorrhage shock, andtraumatic shock, and traumatic brain injury.

The present disclosure also provides a method of treating diseases orconditions of interest including, but are not limited to, T-cellmediated acute or chronic inflammatory diseases or disorders orautoimmune diseases, rheumatoid arthritis, osteoarthritis, systemiclupus erythematosus, Hashimoto's thyroidis, multiple sclerosis,myasthenia gravis, diabetes type I or II and the disorders associatedtherewith, transplant rejection, graft versus host disease, respiratorydiseases, asthma, inflammatory lung injury, inflammatory liver injury,inflammatory glomerular injury, cutaneous manifestations ofimmunologically-mediated disorders or illnesses, inflammatory andhyperproliferative skin diseases, psoriasis, atopic dermatitis, allergiccontact dermatitis, irritant contact dermatitis and further eczematousdermatitises, seborrhoeic dermatitis, inflammatory eye diseases,Sjoegren's syndrome, keratoconjunctivitis, uveitis, inflammatory boweldisease, Crohn's disease or ulcerative colitis, Guillain-Barre syndrome,and allergies.

The subject compounds can be used for treating a cell proliferativedisorder. The present disclosure also provides a method of treatingdiseases or conditions of interest including, but are not limited to,hematopoietic neoplasm, lymphoid neoplasm, T cell neoplasm, Tlymphoblastic leukemia, B cell neoplasm, B-lymphoblastic leukemia,Burkitt's lymphoma, myeloid neoplasm, myeloproferative disease, chronicmyelogenous leukemia (CML), myelodysplastic disease, chronicmyelomonocytic leukemia, myelodysplastic syndrome, and acute myeloidleukemia.

The subject compounds can be used in combination with a prodrug, or asalt thereof, of a Syk kinase inhibitory compound. A Syk kinaseinhibitory compound can have the formula:

wherein

each R³⁰, R³¹ and R³² is independently selected from hydrogen, loweralkyl, lower alkenyl, lower alkynyl, (C₆-C₁₄)aryl, phenyl, 5-14 memberedheteroaryl, (C₇-C₂₀)arylalkyl, benzyl, 7-20 membered heteroarylalkyl,—OR, chloro, fluoro, bromo, cyano, nitro, —C(O)R, —C(O)OR, —NRR,—S(O)₂NRR, —C(O)NRR, —N(R)S(O)₂R and —NC(O)OR, where each R isindependently selected from hydrogen and lower alkyl; and

R^(p) is selected from —CH₂—O—P(O)(OH)₂, —CH₂CH₂—O—P(O)(OH)₂, —CH₂OH.

In combination with a prodrug, or a salt thereof, of a Syk kinaseinhibitory compound, the composition can be used for treating treating acell proliferative disorder. The present disclosure also provides amethod of treating diseases or conditions of interest including, but arenot limited to, hematopoietic neoplasm, lymphoid neoplasm, T cellneoplasm, T lymphoblastic leukemia, B cell neoplasm, B-lymphoblasticleukemia, Burkitt's lymphoma, myeloid neoplasm, myeloproferativedisease, chronic myelogenous leukemia (CML), myelodysplastic disease,chronic myelomonocytic leukemia, myelodysplastic syndrome, and acutemyeloid leukemia.

Since subject compounds possess PKC inhibitory properties, suchcompounds are also useful as research tools. Accordingly, the disclosurealso provides for a method for using a compound of formula (I) or a saltor solvate or stereoisomer thereof as a research tool for studying abiological system or sample, or for discovering new chemical compoundshaving PKC inhibitory properties.

The embodiments are also directed to processes and novel intermediatesuseful for preparing compounds of formula (I) or a salt or solvate orstereoisomer thereof. Accordingly, the present disclosure provides aprocess of preparing a compound of formula (I), the process comprising:

contacting a compound of formula:

with a compound of formula

wherein LG¹ is a leaving group.

In one embodiment, the above process further comprises the step offorming a salt of a compound of formula (I). Embodiments are directed tothe other processes described herein; and to the product prepared by anyof the processes described herein.

The embodiments are also directed to a compound of formula (I) or a saltor solvate or stereoisomer thereof, for use in therapy or as amedicament.

Additionally, the embodiments are directed to the use of a compound offormula (I) or a salt or solvate or stereoisomer thereof, for themanufacture of a medicament; especially for the manufacture of amedicament for the inhibition of protein kinase C(PKC) activity. Theembodiments are also directed to the use of a compound of formula (I) ora salt or solvate or stereoisomer thereof for the manufacture of amedicament for the treatment of a disease or disorder mediated orsustained through the activity of PKC activity. The embodiments are alsodirected to the use of a compound of formula (I) or a salt or solvate orstereoisomer thereof for the manufacture of a medicament for thetreatment of a disease or disorder associated with the activation ofT-cells. Diseases or conditions of interest include, but are not limitedto, an inflammatory disease, an immunological disorder, an autoimmunedisease, an ocular disease or disorder involving inflammatory and/orneovascular events, organ and bone marrow transplant rejection, acute orchronic inflammation, allergies, contact dermatitis, psoriasis,rheumatoid arthritis, multiple sclerosis, type I diabetes, type IIdiabetes, inflammatory bowel disease, Guillain-Barre syndrome, Crohn'sdisease, ulcerative colitis, graft versus host disease, and lupuserythematosus.

The embodiments are also directed to the use of a compound of formula(I) or a salt or solvate or stereoisomer thereof for the manufacture ofa medicament for the treatment of a cell proliferative disorder.Diseases or conditions of interest include, but are not limited to,hematopoietic neoplasm, lymphoid neoplasm, T cell neoplasm, Tlymphoblastic leukemia, B cell neoplasm, B-lymphoblastic leukemia,Burkitt's lymphoma, myeloid neoplasm, myeloproferative disease, chronicmyelogenous leukemia (CML), myelodysplastic disease, chronicmyelomonocytic leukemia, myelodysplastic syndrome, acute myeloidleukemia.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as HPLC, preparativethin layer chromatography, flash column chromatography and ion exchangechromatography. Any suitable stationary phase can be used, includingnormal and reversed phases as well as ionic resins. Most typically thedisclosed compounds are purified via silica gel and/or aluminachromatography. See, e.g., Introduction to Modern Liquid Chromatography,2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons,1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, NewYork, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4.sup.th edition, Vol. 15/1, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

A representative synthesis for compounds of formula (I) is shown inScheme 1.

In Scheme 1, Compound 1-A and Compound 1-B react to form Compound I. Thecompounds react through a nucleophilic aromatic substitution reactionwith the amino group of Compound 1-B and the leaving group (LG¹) ofCompound 1-A. Examples of suitable leaving groups include, but are notlimited to, halogen, mesylate, tosylate, and triflate. The nucleophilicreaction can be run neatly or with a suitable solvent. The nucleophilicreaction can be run at various temperatures, including with cooling, atroom temperature, or with heating. One skilled in the art would be ableto determine suitable reaction conditions according to the specificreactants.

Representative compounds of formula I-A are shown in Scheme 2. In Scheme2, Compound 2-A is an embodiment in which m is two, p is zero and R^(a),R^(b), R^(c) and R^(d) each are methyl. Compound 2-B is an embodiment inwhich m is one, p is one and R^(a), R^(b), R^(c) and R^(d) each aremethyl. Compound 2-C is an embodiment wherein m is two, p is zero, R^(a)and R^(c) are methyl and R^(b) and R^(d) are hydrogen.

Compounds 2-A, 2-B and 2-C can be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. Suitableexemplary methods that may be routinely adapted to synthesize thecompounds are found in Section F of U.S. Patent Publication No.20080306099, the disclosure of which is incorporated herein byreference.

Adapted from Scheme 1 of U.S. Patent Publication No. 20080306099,Compounds 2-A, 2-B and 2-C can be synthesized from substituted orunsubstituted uracils as illustrated in Scheme 3.

In Scheme 3, R¹, R², LG¹, m, and p are as defined herein. According toScheme 3, uracil A-1 is dihalogenated at the 2- and 4-positions using astandard halogenating agent such as POCl₃ (or other standardhalogenating agent) under standard conditions to yield2,4-dichloropyrimidine A-2. Depending upon the R² substituent inpyrimidinediamine A-2, the leaving group at the C4 position is morereactive towards nucleophiles than the leaving group at the C2 position.This differential reactivity can be exploited to synthesize2,4-pyrimidinediamines with different substituents at these positions byfirst reacting 2,4-dichloropyrimidine A-2 with one equivalent of amineA-3, yielding 4N-substituted-2-chloro-4-pyrimidineamine A-4. Furtherreaction of pyrimidineamine A-4 with a second amine nucleophile canyield a 2,4-pyrimidinediamine derivative with different substituents atthe C2 and C4 positions. With continued reference to Scheme 3, A-3compounds wherein m is two, p is zero, R^(a) and R^(c) are methyl andR^(b) and R^(d) are hydrogen are prepared as is known to those of skillin the art and according to the procedures provided by Langlois et al.Eur. J. Med. Chem. 1993, 28, 869-880.

The uracil A-1 starting materials may be purchased from commercialsources or prepared using standard techniques of organic chemistry.Commercially available uracils that can be used as starting materials inScheme 3 include, by way of example and not limitation, uracil (Aldrich#13, 078-8; CAS Registry 66-22-8); 5-bromouracil (Aldrich #85, 247-3;CAS Registry 51-20-7; 5-fluorouracil (Aldrich #85, 847-1; CAS Registry51-21-8); 5-iodouracil (Aldrich #85, 785-8; CAS Registry 696-07-1);5-nitrouracil (Aldrich #85, 276-7; CAS Registry 611-08-5);5-(trifluoromethyl)-uracil (Aldrich #22, 327-1; CAS Registry 54-20-6).Additional 5-substituted uracils are available from GeneralIntermediates of Canada, Inc., Edmonton, Calif. and/or Interchim, Cedex,France, or may be prepared using standard techniques.

A representative synthesis for Compounds 2-B is shown in Scheme 4.

In Scheme 4, R¹, R², and LG¹ are as defined herein. According to Scheme4, the amide of Compound B-1 is reduced. The reduction reaction can becarried out in an ethereal solvent, for example ether ortetrahydrofuran, using lithium aluminium hydride or diborane as reducingagents. Then, Compounds B-2 and B-3 react in a nucleophilic reaction inwhich the amino group displaces one of the leaving groups. Theselectivity of the leaving groups is explained herein. The nucleophilicreaction can be run neatly or with a suitable solvent. The nucleophilicreaction can be run at various temperatures, including with cooling, atroom temperature, or with heating. One skilled in the art would be ableto determine suitable reaction conditions according to the specificreactants.

With continued reference to Scheme 4, amine B-2 has a chiral center.Accordingly, B-2 in the present compounds was used both in racemic andenantiomerically enriched forms. Optically active amine B-2 was preparedas illustrated in Scheme 5 and was incorporated into exemplary compoundsas set forth in Scheme 4.

Representative compounds of formula I-B are shown in Scheme 6. In Scheme6, Compound 3-A is an embodiment in which X¹, X², and X³ are CH; n isone; R³ and R⁴ are hydrogen; and Z¹, Z², and Z³ are CH. Compound 3-B isan embodiment in which X¹, X², and X³ are CH; n is one; R³ and R⁴ arehydrogen; and Z¹, Z², and Z³ are N.

A representative synthesis for Compound 3-A is shown in Scheme 7.

In Scheme 7, LG² is a leaving group. Examples of suitable leaving groupsinclude, but are not limited to, halogen, such as chloride and bromideand sulfonate esters, such as mesylate, tosylate, and triflate.According to Scheme 7, Compound C-1 is treated with2,5-dimethoxytetrahydrofuran and acetic acid to yield Compound C-2.Then, an aromatic formylation is performed on Compound C-2 to yieldCompound C-3. A representative reaction to perform an aromaticformylation uses DMF and POCl₃. Other aromatic formylation reactionsinclude the Reimer-Tiemann reaction using chloroform and a strong baseand the Duff reaction using hexaamine and an acid. Then, Compound C-3undergoes a cyclization reaction where the aldehyde displaces theleaving group LG². The aldehyde of Compound C-3 is reduced and theoxygen nucleophilically displaces the leaving group. The reduction ofthe aldehyde can be performed with various reduction agents, such assodium borohydride, lithium aluminum hydride, sodiumtriacetoxyborohydride, and zinc borohydride. Then, the nitro group ofCompound C-4 is reduced to yield Compound C-5. Suitable reductionmethods to convert the nitro group to an amino group include catalytichydrogenation or reduction agents. Representative methods includecatalytic hydrogenation using platinum oxide, Raney nickel or palladiumhydroxide, iron metal in acidic media, such as refluxing acetic acid orsamarium diiodide.

A representative synthesis for Compounds 3-B is shown in Scheme 8.

According to Scheme 8, the carbonyl of Compound D-1 is converted to athiocarbonyl to yield Compound D-2. The variable groups R³ and R⁴ are asdefined herein with reference to formula (I). In principle any generalthionating reagent can be used to introduce the thiocarbonyl of D-2.Suitable thionating reagents include Lawesson's reagent, Davy's reagent,and Belleau's reagent, and phosphorus pentasulfide. Methods to performthionation reaction can be found in the following references: 1)Shridhar, D. R.; Jogibhukta, M.; Krishnan, V. S. H. IDPL Res. Cent.,Indian Drugs and Pharm. Ltd., Hyderabad, India. Organic Preparations andProcedures International (1984), 16(2), 91-6; 2) Shridhar, D. R.;Jogibhukta, M.; Krishnan, V. S. H. Chem. Div., IDPL Res. Cent.,Hyderabad, India. Indian Journal of Chemistry, Section B: OrganicChemistry Including Medicinal Chemistry (1982), 21B(2), 130-3; and 3)Sastry, C. V. Reddy; Rao, K. Srinivasa; Krishnan, V. S. H.; Rastogi, K.;Jain, M. L.; Narayan, G. K. A. S. S.; Reddi, G. S.; Singh, P. P.; Rao,C. Seshagiri; Junnarkar, A. Y. Chem. Div., Indian Drugs and Pharm. Ltd.,Hyderabad, India. Indian Journal of Chemistry, Section B: OrganicChemistry Including Medicinal Chemistry (1990), 29B(4), 396-8.

Compound D-2 is converted to Compound D-3 through tetrazole syntheticmethods. Reaction of Compound D-2 with an azide with a suitable catalystcan form a tetrazole. Suitable azide compounds for reaction with D-2,include sodium azide and trimethylsilyl azide. Various catalysts can beuse and suitable ones include mercuric acetate, zinc bromide, and zinctriflate. One method of forming tetrazoles such as D-3 can be found inthe following reference: Nelson, Derek W.; Gregg, Robert J.; Kort,Michael E.; Perez-Medrano, Arturo; Voight, Eric A.; Wang, Ying; Grayson,George; Namovic, Marian T.; Donnelly-Roberts, Diana L.; Niforatos,Wende; Honore, Prisca; Jarvis, Michael F.; Faltynek, Connie R.; Carroll,William A. Neuroscience Research, Global Pharmaceutical Research andDevelopment, Abbott Laboratories, Abbott Park, Ill., USA. Journal ofMedicinal Chemistry (2006), 49(12), 3659-3666.

The nitro group of Compound D-3 is reduced to yield Compound D-4.Suitable reduction methods to convert the nitro group to an amino groupinclude catalytic hydrogenation or reduction agents. Representativemethods include catalytic hydrogenation using platinum oxide, Raneynickel or palladium hydroxide, iron metal in refluxing acetic acid, orsamarium diiodide.

Pharmaceutical Compositions

The disclosed compounds are useful, at least, for the inhibition of PKCactivity and the treatment of a disease or disorder that is mediatedthrough the activity of a PKC activity. Accordingly, pharmaceuticalcompositions comprising at least one disclosed compound are alsodescribed herein.

A pharmaceutical composition comprising a subject compound may beadministered to a patient alone, or in combination with othersupplementary active agents. The pharmaceutical compositions may bemanufactured using any of a variety of processes, including, withoutlimitation, conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping, and lyophilizing.The pharmaceutical composition can take any of a variety of formsincluding, without limitation, a sterile solution, suspension, emulsion,lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir orany other dosage form suitable for administration.

A subject compound may be administered to the host using any convenientmeans capable of resulting in the desired reduction in disease conditionor symptom. Thus, a subject compound can be incorporated into a varietyof formulations for therapeutic administration. More particularly, asubject compound can be formulated into pharmaceutical compositions bycombination with appropriate pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

Formulations for pharmaceutical compositions are well known in the art.For example, Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition, 1995, describes exemplaryformulations (and components thereof) suitable for pharmaceuticaldelivery of disclosed compounds. Pharmaceutical compositions comprisingat least one of the subject compounds can be formulated for use in humanor veterinary medicine. Particular formulations of a disclosedpharmaceutical composition may depend, for example, on the mode ofadministration and/or on the location of the infection to be treated. Insome embodiments, formulations include a pharmaceutically acceptablecarrier in addition to at least one active ingredient, such as a subjectcompound. In other embodiments, other medicinal or pharmaceuticalagents, for example, with similar, related or complementary effects onthe affliction being treated can also be included as active ingredientsin a pharmaceutical composition.

Pharmaceutically acceptable carriers useful for the disclosed methodsand compositions are conventional in the art. The nature of apharmaceutical carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can optionally contain minor amounts ofnon-toxic auxiliary substances (e.g., excipients), such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like;for example, sodium acetate or sorbitan monolaurate. Other non-limitingexcipients include, nonionic solubilizers, such as cremophor, orproteins, such as human serum albumin or plasma preparations.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

The disclosed pharmaceutical compositions may be formulated as apharmaceutically acceptable salt of a disclosed compound.Pharmaceutically acceptable salts are non-toxic salts of a free baseform of a compound that possesses the desired pharmacological activityof the free base. These salts may be derived from inorganic or organicacids. Non-limiting examples of suitable inorganic acids arehydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid,hydroiodic acid, and phosphoric acid. Non-limiting examples of suitableorganic acids are acetic acid, propionic acid, glycolic acid, lacticacid, pyruvic acid, malonic acid, succinic acid, malic acid, maleicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, methyl sulfonic acid, salicylic acid, formicacid, trichloroacetic acid, trifluoroacetic acid, gluconic acid,asparagic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonicacid, naphthalenesulfonic acid, and the like. Lists of other suitablepharmaceutically acceptable salts are found in Remington'sPharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton,Pa., 1985. A pharmaceutically acceptable salt may also serve to adjustthe osmotic pressure of the composition.

A subject compound can be used alone or in combination with appropriateadditives to make tablets, powders, granules or capsules, for example,with conventional additives, such as lactose, mannitol, corn starch orpotato starch; with binders, such as crystalline cellulose, cellulosederivatives, acacia, corn starch or gelatins; with disintegrators, suchas corn starch, potato starch or sodium carboxymethylcellulose; withlubricants, such as talc or magnesium stearate; and if desired, withdiluents, buffering agents, moistening agents, preservatives andflavoring agents. Such preparations can be used for oral administration.

A subject compound can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives. The preparation may also be emulsified or the activeingredient encapsulated in liposome vehicles. Formulations suitable forinjection can be administered by an intravitreal, intraocular,intramuscular, subcutaneous, sublingual, or other route ofadministration, e.g., injection into the gum tissue or other oraltissue. Such formulations are also suitable for topical administration.

In some embodiments, a subject compound can be delivered by a continuousdelivery system. The term “continuous delivery system” is usedinterchangeably herein with “controlled delivery system” and encompassescontinuous (e.g., controlled) delivery devices (e.g., pumps) incombination with catheters, injection devices, and the like, a widevariety of which are known in the art.

A subject compound can be utilized in aerosol formulation to beadministered via inhalation. A subject compound can be formulated intopressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, a subject compound can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. A subject compound can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectcompound calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for a subject compound depend onthe particular compound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

The dosage form of a disclosed pharmaceutical composition will bedetermined by the mode of administration chosen. For example, inaddition to injectable fluids, topical or oral dosage forms may beemployed. Topical preparations may include eye drops, ointments, spraysand the like. Oral formulations may be liquid (e.g., syrups, solutionsor suspensions), or solid (e.g., powders, pills, tablets, or capsules).Methods of preparing such dosage forms are known, or will be apparent,to those skilled in the art.

Certain embodiments of the pharmaceutical compositions comprising asubject compound may be formulated in unit dosage form suitable forindividual administration of precise dosages. The amount of activeingredient administered will depend on the subject being treated, theseverity of the affliction, and the manner of administration, and isknown to those skilled in the art. Within these bounds, the formulationto be administered will contain a quantity of the extracts or compoundsdisclosed herein in an amount effective to achieve the desired effect inthe subject being treated.

Each therapeutic compound can independently be in any dosage form, suchas those described herein, and can also be administered in various ways,as described herein. For example, the compounds may be formulatedtogether, in a single dosage unit (that is, combined together in oneform such as capsule, tablet, powder, or liquid, etc.) as a combinationproduct. Alternatively, when not formulated together in a single dosageunit, an individual subject compound may be administered at the sametime as another therapeutic compound or sequentially, in any orderthereof.

The subject compound can be administered in combination with anotherprotein kinase inhibitor. For example, the subject compound can beadministered with a Syk kinase inhibitor. Various compounds that inhibitSyk kinase or Syk/Flt-3 kinase activity can be used in the methodsdescribed herein. These include, among others, small organic molecules,peptides or proteins, or nucleic acids. As used herein, a “Sykinhibitor” or “Syk kinase inhibitory compound” refers to any compoundthat directly inhibits the activity of Syk kinase itself or inhibits Sykinteraction with other cellular targets needed for proper Syk functionin the IC₅₀ range described herein. Inhibitors as used herein includethe classical description of enzyme inhibitors, such as competitive,noncompetitive and uncompetitive inhibitors, and thus encompassescompounds that inhibit Syk kinase activity by, for example, binding toSyk kinase so as to inhibit access of a substrate to an active site,binding to Syk kinase so as to distort the active site to reduce bindingto substrate, and/or bind a Syk kinase-substrate complex. Compounds thatare Syk inhibitors are generally those that display an IC₅₀ with respectto a Syk kinase activity, such as the ability of Syk kinase tophosphorylate a synthetic or endogenous substrate, in an in vitro orcellular assay, in the range of about 5 μM or lower, about 1 μM orlower, about 500 nm or lower, about 100 nM or lower, about 50 nM orlower, about 10 mM or lower, or about 1 nM or lower. For instance,exemplary Syk inhibitor compounds are disclosed in U.S. application Ser.No. 10/631,029 and PCT publication WO 2004/014382. Both U.S. applicationSer. No. 10/631,029 and PCT publication WO 2004/014382 are incorporatedherein by reference. Skilled artisans will appreciate that compoundsexhibiting lower IC₅₀, such as in the range of about 100 nM, 10 nM, 1nM, or even lower, are useful for the methods herein.

Methods of Administration

The subject compounds can inhibit a protein kinase C activity.Accordingly, the subject compounds are useful for treating a disease ordisorder that is mediated through the activity of a PKC activity in asubject. Accordingly, the subject compounds are useful for treating adisease or disorder that is associated with the activation of T-cells ina subject.

The route of administration will be selected according to a variety offactors including, but not necessarily limited to, the condition to betreated, the formulation and/or device used, the patient to be treated,and the like. Routes of administration useful in the disclosed methodsinclude but are not limited to oral and parenteral routes, such asintravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic,nasal, and transdermal. Formulations for these dosage forms aredescribed herein.

An effective amount of a subject compound will depend, at least, on theparticular method of use, the subject being treated, the severity of theaffliction, and the manner of administration of the therapeuticcomposition. A “therapeutically effective amount” of a composition is aquantity of a specified compound sufficient to achieve a desired effectin a subject (host) being treated. For example, this may be the amountof a subject compound necessary to prevent, inhibit, reduce or relieve adisease or disorder that is mediated through the activity of a PKCactivity in a subject. Ideally, a therapeutically effective amount of acompound is an amount sufficient to prevent, inhibit, reduce or relievea disease or disorder that is mediated through the activity of a PKCactivity in a subject without causing a substantial cytotoxic effect onhost cells.

Therapeutically effective doses (or growth inhibitory amounts) of asubject compound or pharmaceutical composition can be determined by oneof skill in the art, with a goal of achieving local (e.g., tissue)concentrations that are at least as high as the IC₅₀ of an applicablecompound disclosed herein.

An example of a dosage range is from about 0.1 to about 200 mg/kg bodyweight orally in single or divided doses. In particular examples, adosage range is from about 1.0 to about 100 mg/kg body weight orally insingle or divided doses, including from about 1.0 to about 50 mg/kg bodyweight, from about 1.0 to about 25 mg/kg body weight, from about 1.0 toabout 10 mg/kg body weight (assuming an average body weight ofapproximately 70 kg; values adjusted accordingly for persons weighingmore or less than average). For oral administration, the compositionsare, for example, provided in the form of a tablet containing from about50 to about 1000 mg of the active ingredient, particularly about 75 mg,about 100 mg, about 200 mg, about 400 mg, about 500 mg, about 600 mg,about 750 mg, or about 1000 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 500 mgto about 1000 mg active ingredient is administered once (e.g., a loadingdose) followed by administration of ½ dosage tablets (e.g., from about250 to about 500 mg) each 6 to 24 hours for at least 3 days.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the subject compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex and diet of the subject, mode and time of administration,rate of excretion, drug combination, and severity of the condition ofthe host undergoing therapy.

The present disclosure also contemplates combinations of one or moredisclosed compounds with one or more other agents or therapies useful inthe treatment of a disease or disorder. In certain instances, thedisease or disorder is mediated through the activity of a PKC activityin a subject. In certain instances, the disease or disorder is cellproliferative disorder. For example, one or more disclosed compounds maybe administered in combination with effective doses of other medicinaland pharmaceutical agents, or in combination other non-medicinaltherapies, such as hormone or radiation therapy. The term“administration in combination with” refers to both concurrent andsequential administration of the active agents.

Protein Kinase C

Protein Kinase C

PKC is a family of enzymes that function as serine/threonine kinases.The isoenzymes of PKC differ in their tissue distribution, enzymaticselectivity, requirement for Ca²⁺, and regulation. PKCs play animportant role in cell-cell signaling, gene expression and in thecontrol of cell differentiation and growth.

The subject compound can be a selective inhibitor of PKC, e.g. aninhibitor selective for PKC over one or more other protein kinases, e.g.over one or more tyrosine kinases, for instance, over one or morenon-receptor or receptor tyrosine kinases, e.g. over one or more of PKA,PKB, Abl Met, Src, Ins-R, Flt-3, JAK-2, KDR and/or Ret proteins. Theselective PKC inhibitors may optionally be selective over one or moreserine/threonine kinases, e.g. one or more serine/threonine kinaseswhich do not belong to the CDK family. The subject compounds can exhibita selectivity of at least 10 fold, or 20 fold, or 100 fold for the PKCover one or more other protein kinases, e.g. over one or more tyrosinekinases, e.g. over Flt-3, JAK-2, KDR and/or Ret proteins, or over one ormore serine/threonine kinases which do not belong to the CDK family.

The selectivity of a selective inhibitor of PKC over other proteinkinases may be calculated as the ratio of the IC₅₀ measured for PKC inan assay described herein over the IC₅₀ determined for another kinase.In a certain instance, there is provided a PKC inhibitor for which theratio of the IC₅₀ value as determined in an Allogeneic Mixed LymphocyteReaction (MLR) assay to the IC₅₀ value as determined in a BM assay ishigher than 5, 10, 20, or 30. MLR and BM assays can be done according toknown methods, e.g. mouse or human MLR and BM assays, such as disclosedherein.

The disclosure provides an inhibitor of PKC, which can be anisozyme-selective PKC inhibitor, wherein the subject compound possessesselectivity for the isoforms θ and α of PKC over one or more of theother PKC isoforms. In a certain instance, the subject compoundpossesses selectivity for the isoform θ of PKC over one or more of theother PKC isoforms. In a certain instance, the subject compoundpossesses selectivity for the isoform α of PKC over one or more of theother PKC isoforms. In one embodiment, the disclosed compounds exhibitselectivity for PKC θ and PKC α over at least one PKC isoform.

A subject compound can show a selectivity of at least 10 fold, or 20fold, or 100 fold for the isoforms θ or α of PKC over one or more of theother PKC isoforms. Selectivity for the isoforms θ or α of PKC over oneor more of the other PKC isoforms can be measured by comparing the IC₅₀of the subject compound for the isoforms θ or α of PKC to the IC₅₀ ofthe subject compound for the other PKC isoforms. In a certain instance,the selectivity can be determined by calculating the ratio of IC₅₀ ofthe subject compound for the other isoforms of PKC to the IC₅₀ of thesubject compound for θ or α isoforms of PKC. In certain examples subjectcompounds exhibit a selectivity for PKC θ, α or both over another PKCisoform of at least about 2-fold, such as from about 3-fold to about300-fold, from about 10-fold to about 100-fold or from about 5-fold to50-fold. IC₅₀ values are obtained, for example, according to PKC assaysdescribed herein. The subject compounds can show an IC₅₀ value for theisoforms θ or α of PKC of 1 μM or less, such as less than about 300 nM,such as from about 1 nM to about 250 nM, less than 100 nM or even lessthan 10 nM in the assays disclosed herein.

The subject compounds can show a selectivity of the isoforms θ or μ ofPKC over other isoforms of PKC, as well as a selectivity over one ormore of the other protein kinases, e.g. over one or more tyrosinekinases, or over one or more serine/threonine kinases which do notbelong to the CDK-family, e.g. over one or more of PKA, PKB, Abl, Met,Src, Ins-it, Flt-3, JAK-2, KDR and Ret proteins, e.g. over one or moreof Flt-3, JAK-2, KDR and Ret proteins.

Certain isozymes of PKC have been implicated in the mechanisms ofvarious disease states, including, but not necessarily limited to, thefollowing: cancer (PKC α, βI, βII, and δ); cardiac hypertrophy and heartfailure (PKC βI and PKC βII) nociception (PKC γ and ε); ischemiaincluding myocardial infarction (PKC ε and δ); immune response,particularly T-cell mediated (PKC θ and α); and fibroblast growth andmemory (PKC δ and ζ). The role of PKC ε is also implicated in painperception. PKC inhibitors can also be used for treating an oculardisease or disorder involving inflammatory and/or neovascular events.

The subject compounds can be used in the treatment of mammalian(especially human) disease states characterized by aberrant, elevatedactivity of a PKC isozyme in a tissue as compared to non-disease tissueof the same origin. PKC isozymes and disease states and/or biologicalfunctions amenable to therapy by inhibition of activity of the PKCisozyme include, but are not necessarily limited to: PKC α(hyperproliferative cellular diseases, such as cancer); PKC βI and PKCβII (cardiac hypertrophy and heart failure); PKC γ (pain management);PKC δ (ischemia, hypoxia (e.g., such as in myocardial infarction and instroke); apoptosis induced by UV irradiation; and aberrant fibroblastgrowth (e.g., as may occur in wound healing)); PKC ε (pain management,myocardial dysfunction); PKC θ (immune system diseases, particularlythose involving T-cell mediated responses); and PKC ζ (memory andfibroblast growth).

PKC Theta

PKC θ is expressed predominantly in lymphoid tissue and skeletal muscle.PKC θ is selectively expressed in T-cells and plays a role in matureT-cell activation. It has been shown that PKC θ is involved in T-cellreceptor (TCR)-mediated T-cell activation but inessential duringTCR-dependent thymocyte development. PKC θ, but not other PKC isoforms,translocates to the site of cell contact between antigen-specificT-cells and antigen presenting cells (APC), where it localizes with theTCR in the central core of the T-cell activation. PKC θ, but not the α,ε, or ζ isoenzymes, can selectively activate a FasL promoter-reportergene and upregulate the mRNA or cell surface expression of endogenousFasL. On the other hand, PKC θ and ε can promote T-cell survival byprotecting the cells from Fas-induced apoptosis, and this protectiveeffect was mediated by promoting p90Rsk-dependent phosphorylation ofBCL-2 family member BAD. Thus, PKC θ appears to play a dual regulatoryrole in T-cell apoptosis.

PKC θ inhibitors can find use in the treatment or prevention ofdisorders or diseases mediated by T lymphocytes, for example, autoimmunedisease such as rheumatoid arthritis, psoriasis and lupus erythematosus,and inflammatory disease such as asthma and inflammatory bowel diseases.

PKC θ is a drug target for immunosuppression in transplantation andautoimmune diseases (Isakov et al. (2002) Annual Review of Immunology,20, 761-794). PCT Publication WO2004/043386 identifies PKC θ as a targetfor treatment of transplant rejection and multiple sclerosis. PKC θ alsoplays a role in inflammatory bowel disease (The Journal of Pharmacologyand Experimental Therapeutics (2005), 313 (3), 962-982), asthma (WO2005062918), and lupus (Current Drug Targets: Inflammation & Allergy(2005), 4 (3), 295-298).

In addition, PKC θ is highly expressed in gastrointestinal stromaltumors (Blay, P. et al. (2004) Clinical Cancer Research, 10, 12, Pt. 1),it has been suggested that PKC θ is a molecular target for treatment ofgastrointestinal cancer (Wiedmann, M. et al. (2005) Current Cancer DrugTargets 5(3), 171).

Experiments induced in PKC θ knock-out mice led to the conclusion thatPKC θ inactivation prevented fat-induced defects in insulin signallingand glucose transport in skeletal muscle (Kim J. et al, 2004, The J. ofClinical Investigation 114 (6), 823). This data indicates PKC θ is atherapeutic target for the treatment of type 2 diabetes, and hence PKC θinhibitors can be useful for treating such disease.

Syk Kinase

As disclosed herein, the subject compound can be administered incombination with another protein kinase inhibitor, such as a Syk kinaseinhibitor.

“Syk” or “Syk kinase” refers to the 72 kDa non-receptor (cytoplasmic)spleen protein tyrosine kinase expressed in B-cells and otherhematopoetic cells. Syk kinase is characterized by two consensusSrc-homology 2 (SH2) domains in tandem that bind to phosphorylatedimmunoreceptor tyrosine-based activation motifs (“ITAMs”), a “linker”domain and a catalytic domain (for a review, see Sada et al., 2001, J.Biochem. (Tokyo) 130:177-186 and also Turner et al., 2000, ImmunologyToday 21:148-154 and Wong et al., 2004, Expert Opin Investig Drugs13(7):743-62). Syk kinase is also critical for tyrosine phosphorylationof multiple proteins which regulate important pathways leading fromimmunoreceptors, such as Ca²⁺ mobilization and mitogen-activated proteinkinase (MAPK) cascades and degranulation. Syk kinase also plays acritical role in integrin signaling in neutrophils (see, e.g., Mocsai etal. 2002, Immunity 16:547-558). Syk kinase includes kinases from anyspecies of animal, including but not limited to, homo sapiens, simian,bovine, porcine, rodent, etc., recognized as belonging to the Sykfamily. Specifically included are isoforms, splice variants, allelicvariants, mutants, both naturally occurring and man-made. The amino acidsequences of such Syk kinases are available from GENBANK. Specificexamples of mRNAs encoding different isoforms of human Syk kinase areavailable at GENBANK accession no. gi|21361552|ref|_(NM)—003177.2,gi|496899|emb|Z29630.1|HSSYKPTK[496899] andgi|15030258|gb|BC011399.1|BC011399[15030258], which are incorporatedherein by reference.

Therapeutic Applications

The subject compounds are useful for treating a disease or disorder thatis mediated through, or exacerbated by, the activity of a PKC in asubject in need of treatment. Also, the compounds are useful fortreating a disease or disorder that is associated with aberrant orotherwise undesirable T cell activation in a subject.

Accordingly, the present disclosure provides methods of treating aninflammatory disease in a subject by administering an effective amountof a subject compound, including a salt or solvate or stereoisomerthereof, so as to treat inflammation. Inflammatory diseases contemplatedfor therapy include acute and chronic inflammation mediated orexacerbated by PKC activity

The present disclosure also provides methods of treating an autoimmunedisease in a subject by administering to the subject an effective amountof a subject compound, including a salt or solvate or stereoisomerthereof, so as to treat the autoimmune disease.

The present disclosure also provides methods of treating an oculardisease or disorder involving inflammatory and/or neovascular events byadministration of a subject compound, including a salt or solvate orstereoisomer thereof, in an effective amount.

Diseases or conditions of interest for treatment according to thepresent disclosure include, but are not limited to, atherosclerosis,vascular occlusion due to vascular injury such as angioplasty,restenosis, obesity, syndrome X, impaired glucose tolerance, polycysticovary syndrome, hypertension, heart failure, chronic obstructivepulmonary disease, CNS diseases such as Alzheimer disease or amyotrophiclateral sclerosis, cancer, infectious diseases such as: AIDS, septicshock or adult respiratory distress syndrome, ischemia/reperfusioninjury, e.g.: myocardial infarction, stroke, gut ischemia, renal failureor hemorrhage shock, and traumatic shock, e.g. traumatic brain injury.

Further diseases or conditions of interest for treatment according tothe present disclosure include, but are not limited to, T-cell mediatedacute or chronic inflammatory diseases or disorders or autoimmunediseases, rheumatoid arthritis, osteoarthritis, systemic lupuserythematosus, Hashimoto's thyroidis, multiple sclerosis, myastheniagravis, diabetes type I or II and the disorders associated therewith,transplant rejection, graft versus host disease, respiratory diseases,asthma, inflammatory lung injury, inflammatory liver injury,inflammatory glomerular injury, cutaneous manifestations ofimmunologically-mediated disorders or illnesses, inflammatory andhyperproliferative skin diseases (such as psoriasis, atopic dermatitis,allergic contact dermatitis, irritant contact dermatitis and furthereczematous dermatitises, seborrhoeic dermatitis), inflammatory eyediseases (such as Sjoegren's syndrome, keratoconjunctivitis, uveitis)inflammatory bowel disease, Crohn's disease or ulcerative colitis,Guillain-Barre syndrome, and allergies.

The subject compounds can also be used for preventing or treating ordelaying ocular diseases and disorders involving inflammation and/orneovascularization. Ocular diseases or disorders involving inflammatoryand/or neovascular events include, but are not limited to, maculardegeneration (AMD), diabetic ocular diseases or disorders, uveitis,optic neuritis, ocular edema, ocular angiogenesis, ischemic retinopathy,anterior ischemic optic neuropathy, optic neuropathy and neuritis,macular edema, cystoid macular edema (CME), retinal disease or disorder,such as retinal detachment, retinitis pigmentosa (RP), Stargart'sdisease, Best's vitelliform retinal degeneration, Leber's congenitalamaurosis and other hereditary retinal degenerations, Sorsby's fundusdystrophy, pathologic myopia, retinopathy of prematurity (ROP), Leber'shereditary optic neuropathy, corneal transplantation or refractivecorneal surgery, keratoconjunctivitis, or dry eye.

Generally, cell proliferative disorders treatable with the subjectcompound disclosed herein relate to any disorder characterized byaberrant cell proliferation. These include various tumors and cancers,benign or malignant, metastatic or non-metastatic. Specific propertiesof cancers, such as tissue invasiveness or metastasis, can be targetedusing the methods described herein. Cell proliferative disorders includea variety of cancers, including, among others, breast cancer, ovariancancer, renal cancer, gastrointestinal cancer, kidney cancer, bladdercancer, pancreatic cancer, lung squamous carcinoma, and adenocarcinoma.

In some embodiments, the cell proliferative disorder treated is ahematopoietic neoplasm, which is aberrant growth of cells of thehematopoietic system. Hematopoietic malignancies can have its origins inpluripotent stem cells, multipotent progenitor cells, oligopotentcommitted progenitor cells, precursor cells, and terminallydifferentiated cells involved in hematopoiesis. Some hematologicalmalignancies are believed to arise from hematopoietic stem cells, whichhave the ability for self renewal. For instance, cells capable ofdeveloping specific subtypes of acute myeloid leukemia (AML) upontransplantation display the cell surface markers of hematopoietic stemcells, implicating hematopoietic stem cells as the source of leukemiccells. Blast cells that do not have a cell marker characteristic ofhematopoietic stem cells appear to be incapable of establishing tumorsupon transplantation (Blaire et al., 1997, Blood 89:3104-3112). The stemcell origin of certain hematological malignancies also finds support inthe observation that specific chromosomal abnormalities associated withparticular types of leukemia can be found in normal cells ofhematopoietic lineage as well as leukemic blast cells. For instance, thereciprocal translocation t(9q34;22q11) associated with approximately 95%of chronic myelogenous leukemia appears to be present in cells of themyeloid, erythroid, and lymphoid lineage, suggesting that thechromosomal aberration originates in hematopoietic stem cells. Asubgroup of cells in certain types of CML displays the cell markerphenotype of hematopoietic stem cells.

Although hematopoietic neoplasms often originate from stem cells,committed progenitor cells or more terminally differentiated cells of adevelopmental lineage can also be the source of some leukemias. Forexample, forced expression of the fusion protein Bcr/Abl (associatedwith chronic myelogenous leukemia) in common myeloid progenitor orgranulocyte/macrophage progenitor cells produces a leukemic-likecondition. Moreover, some chromosomal aberrations associated withsubtypes of leukemia are not found in the cell population with a markerphenotype of hematopoietic stem cells, but are found in a cellpopulation displaying markers of a more differentiated state of thehematopoietic pathway (Turhan et al., 1995, Blood 85:2154-2161). Thus,while committed progenitor cells and other differentiated cells may haveonly a limited potential for cell division, leukemic cells may haveacquired the ability to grow unregulated, in some instances mimickingthe self-renewal characteristics of hematopoietic stem cells (Passegueet al., Proc. Natl. Acad. Sci. USA, 2003, 100:11842-9).

In some embodiments, the hematopoietic neoplasm treated is a lymphoidneoplasm, where the abnormal cells are derived from and/or display thecharacteristic phenotype of cells of the lymphoid lineage. Lymphoidneoplasms can be subdivided into B-cell neoplasms, T and NK-cellneoplasms, and Hodgkin's lymphoma. B-cell neoplasms can be furthersubdivided into precursor B-cell neoplasm and mature/peripheral B-cellneoplasm. Exemplary B-cell neoplasms are precursor B-lymphoblasticleukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia) whileexemplary mature/peripheral B-cell neoplasms are B-cell chroniclymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-celllymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma,extranodal marginal zone B-cell lymphoma of MALT type, nodal marginalzone B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, diffuselarge B-cell lymphoma, mediastinal large B-cell lymphoma, primaryeffusion lymphoma, and Burkitt's lymphoma/Burkitt cell leukemia. T-celland Nk-cell neoplasms are further subdivided into precursor T-cellneoplasm and mature (peripheral) T-cell neoplasms. Exemplary precursorT-cell neoplasm is precursor T-lymphoblastic lymphoma/leukemia(precursor T-cell acute lymphoblastic leukemia) while exemplary mature(peripheral) T-cell neoplasms are T-cell prolymphocytic leukemia T-cellgranular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-celllymphoma/leukemia (HTLV-1), extranodal NK/T-cell lymphoma, nasal type,enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, Mycosisfungoides/Sezary syndrome, Anaplastic large-cell lymphoma, T/null cell,primary cutaneous type, Peripheral T-cell lymphoma, not otherwisecharacterized, Angioimmunoblastic T-cell lymphoma, Anaplastic large-celllymphoma, T/null cell, primary systemic type. The third member oflymphoid neoplasms is Hodgkin's lymphoma, also referred to as Hodgkin'sdisease. Exemplary diagnosis of this class that can be treated with thecompounds include, among others, nodular lymphocyte-predominantHodgkin's lymphoma, and various classical forms of Hodgkin's disease,exemplary members of which are Nodular sclerosis Hodgkin's lymphoma(grades 1 and 2), Lymphocyte-rich classical Hodgkin's lymphoma, Mixedcellularity Hodgkin's lymphoma, and Lymphocyte depletion Hodgkin'slymphoma.

In some embodiments, the hematopoietic neoplasm treated is a myeloidneoplasm. This group comprises a large class of cell proliferativedisorders involving or displaying the characteristic phenotype of thecells of the myeloid lineage. Myeloid neoplasms can be subdivided intomyeloproliferative diseases, myelodysplastic/myeloproliferativediseases, myelodysplastic syndromes, and acute myeloid leukemias.Exemplary myeloproliferative diseases are chronic myelogenous leukemia(e.g., Philadelphia chromosome positive (t(9;22)(qq34;q11)), chronicneutrophilic leukemia, chronic eosinophilic leukemialhypereosinophilicsyndrome, chronic idiopathic myelofibrosis, polycythemia vera, andessential thrombocythemia. Exemplary myelodysplastic/myeloproliferativediseases are chronic myelomonocytic leukemia, atypical chronicmyelogenous leukemia, and juvenile myelomonocytic leukemia. Exemplarymyelodysplastic syndromes are refractory anemia, with ringedsideroblasts and without ringed sideroblasts, refractory cytopenia(myelodysplastic syndrome) with multilineage dysplasia, refractoryanemia (myelodysplastic syndrome) with excess blasts, 5q-syndrome, andmyelodysplastic syndrome with t(9;12)(q22;p12) (TEL-Syk fusion; see,e.g., Kuno et al., 2001, Blood 97:1050).

In some embodiments, the composition can be used to treat acute myeloidleukemias (AML), which represent a large class of myeloid neoplasmshaving its own subdivision of disorders. These subdivisions include,among others, AMLs with recurrent cytogenetic translocations, AML withmultilineage dysplasia, and other AML not otherwise categorized.Exemplary AMLs with recurrent cytogenetic translocations include, amongothers, AML with t(8;21)(q22;q22), AML1(CBF-alpha)/ETO, Acutepromyelocytic leukemia (AML with t(15;17)(q22;q11-12) and variants,PML/RAR-alpha), AML with abnormal bone marrow eosinophils(inv(16)(p13q22) or t(16;16)(p13;q11), CBFb/MYH11X), and AML with 11q23(MLL) abnormalities. Exemplary AML with multilineage dysplasia are thosethat are associated with or without prior myelodysplastic syndrome.Other acute myeloid leukemias not classified within any definable groupinclude, AML minimally differentiated, AML without maturation, AML withmaturation, Acute myelomonocytic leukemia, Acute monocytic leukemia,Acute erythroid leukemia, Acute megakaryocytic leukemia, Acutebasophilic leukemia, and Acute panmyelosis with myelofibrosis.

In other aspects, cell proliferative disorders comprise virally mediatedtumors. These can arise from infection of cells by an oncogenic virusthat has the capability of transforming a normal cell into a tumor cell.Because rates of viral infection far exceed the number of actualincidence of cell transformation, viral mediated transformationgenerally act together with other cellular factors to generate atransformed tumor cell. Thus, a virally mediated tumor does not requirethe virus to be the sole causative agent of the cell proliferativedisorder, but rather that the viral infection or persistent presence ofvirus is associated with the generation of the tumor. Generally, tumorswhere the causative agent is a virus typically has continual expressionof a limited number of viral genes and that viral these oncogenes,expressed as part of the viral infection or through persistence of thevirus, disrupts the normal cellular gene expression and signaltransduction pathways. Without being bound by theory, viral oncogenesinvolved in cell transformation appear to disrupt four main cellularprocesses: cell surface receptors that interact with growth factors andextracellular matrix, transmembrane signaling networks, cytosolicelements such as soluble proteins and second messengers, and nuclearproteins including DNA binding proteins and factors which functiondirectly and indirectly in gene regulation and replication.

In some embodiments, the virally mediated tumor treatable with thesubject compound disclosed herein is associated with any virus thatencodes an immunoreceptor tyrosine-based activation motif (ITAM) capableof modulating Syk activity. This motif, as noted above, refers to aconserved amino acid sequence motif that functions by interacting withand activating nonreceptor tyrosine kinases. ITAM motifs are found in,among others, the p and y chains of FcεRI, the c subunit of the T cellreceptor, and immunoglobulin β (Igβ) and Igα of the B cell receptor. Thecanonical sequence motif is typically Yxx(L/I)x.sub.6-8Yxx(L/I), where xrepresents any amino acid. Generally, the tyrosine residues in the motifare involved in ITAM signaling and are substrates for phosphorylation bySrc family of kinases. The phosphorylated form of ITAMs function asinteraction sites for SH2 (src homology domain) containing signalingproteins, such as Syk/ZAP-70 kinases. In addition to its presence in avariety of cellular cell surface molecules, the ITAM sequences have beenidentified in virally encoded proteins. In view of the descriptionsherein indicating function of Syk kinase as an oncogene, tumorsassociated with viruses carrying genes encoding proteins with ITAMsequences can be treated with Syk inhibitor compounds.

Accordingly, in some embodiments, the virally mediated tumor treatablewith the subject compounds is associated with Kaposi's sarcoma (KS)associated herpes virus, a lymphotropic virus implicated in Kaposi'ssarcoma, a rare malignancy found at higher incidence among HIV infectedpopulation. The KS associated herpes virus encodes a transmembraneprotein termed KI having an immunoreceptor tyrosine-based activationmotif (ITAM)-like sequence. The KI gene product is thought to act in aconstitutive manner through its cysteine-rich ectodomain to activate Sykand its related kinase Zap-70 (Lagunoff, M. et al., 1999, Proc. Natl.Acad. Sci. USA 96(10):5704-5709). In further support of the methodsherein, transgenic mice bearing the KI gene appears to increase theincidence of certain sarcomas and lymphomas in an infected animal,indicating a role for KI activity in oncogenesis (Prakash et al., 2002,J. Natl. Cancer Inst. 94:926-35).

In some embodiments, the virally mediated tumor is associated withEpstein Barr Virus (EBV). Epstein Barr Virus is a member of theHerpesviridae family that, following primary infection, replicates inthe epithelial cells of the oropharynx and infect recirculating Blymphocytes. Infection can lead to acute infectious mononucleosis, alsoknown as glandular fever. Infectious mononucleosis is a benignlymphoproliferative disease characterized by transient immunosuppressionand an expansion of atypical lymphocytes, the majority of which are CD8⁺^(T) cells. In these T cells, EBV establishes a latent but persistentinfection during which a select set of viral genes are expressed. Theentire genome can persist in the proliferating lymphocytes as episomalDNA. EBV infection is associated with Burkitt's lymphoma, Hodgkin'slymphoma, and adult T cell leukemia.

The LMP2A protein encoded by the EBV genome is a transmembrane proteinthought to play a role in maintaining the latency of the EBV virusfollowing infection. It consists of an extended amino terminal tail, 12membrane spanning domains, and a cytoplasmic domain. The amino terminalregion contains the ITAM motif, which allows interaction of LMP2A withSyk kinase (Fruehling et al., 1997, Virology, 235:241-251). LMP2Aappears to regulate Syk kinase in lymphoid cells to promote B-cellsurvival and maintain latency. Because Syk plays a role in the signaltransduction pathways that regulate other signaling pathways, such asPI-3K, BLNK, and phospholipase y2 and is involved in enhancing lymphoidcell survival, improper Syk activation through LMP2A protein or othervirally mediated effectors may play a role in inducing aberrantlymphoproliferation (Caldwell et al., 2000, J Virol 74(19):9115;Caldwell et al., 1998, Immunity 9:405)).

In some embodiments, the virally mediated tumor to be treated with thesubject composition is associated with Human T-cell Lymphotropic Virus(HTLV-1 virus), a retrovirus in the same class of virus as the AIDSvirus, HIV-1. The virus is tropic for CD4⁺ ^(T) -cells although CD8⁺^(T) -cells can also serve as a viral reservoir. HTLV-1 infection isassociated with, among others, adult T-cell Leukemia/lymphoma (ATLL) anda number of other lymphocyte disorders. During HTLV-1 infection, Syk isexpressed in infected cells while expression of the Syk related kinase,ZAP-70, is absent (Weil et al., 1999, J. Virol. 73(5):3709-17).Dysregulation of a number of kinases, including Syk, is implicated inHTLV-1 mediated induction of adult T-cell leukemia.

In some embodiments, the virally mediated tumor is associated withmammary tumor virus (MTV). ITAM sequences are found within the Env geneof murine mammary tumor virus (MMTV), a B type retrovirus identified asan etiological agent for breast cancer in mice. Mouse mammary epithelialcells transfected with MMTV Env gene display characteristics of atransformed phenotype, such as colony formation in soft agar andinvasiveness in basement membrane preparations (Katz et al., 2005, J ExpMed. 201(3):431-9). Murine mammary tumor virus-like sequences are alsopresent in human cancers, such as breast cancer and T cell lymphomas(Wang et al., 2000, Clinical Cancer Res. 6:1273-1278), and correlatedwith tumorigenesis as these sequences are not observed in the majorityof normal breast tissue.

It is to be understood that use of subject composition for treatingvirally mediated tumors is not limited to tumors associated with theviruses specified above. As noted, any tumors associated with anoncogenic virus in which Syk is activated as part of its oncogenicmechanism, whether or not it involves ITAM sequences, can be targetedusing the subject compounds.

Characterization of Functional Properties

The following are exemplary assays useful in characterizing activitiesof a compound of interest.

A. In Vitro

1. Protein Kinase C Assay

The inhibition of PKC activity was measured by monitoring the productionof phosphorylated peptide by fluorescence polarization at differentconcentrations of the inhibitor. Reactions were carried out in 96-wellplate format with a total volume of 20 μL containing 20 mM HEPES, pH7.4, 5 mM MgCl₂, 0.2 mM CaCl₂, 1 mM DTT, 0.02% Brij-35, 0.1 mg/mLphosphatidylserine, 0.02 mg/mL dioleoyl-sn-glycerol and 5 μM each of ATPand the peptide substrate. Compounds were first diluted serially in DMSOand then transferred to a solution containing the above concentrationsof HEPES, MgCl₂, CaCl₂, DTT, and Brij-35 to yield 5× compound solutionsin 2% DMSO, which was then added to the reaction solution. Reactionswere initiated by the addition of PKC at a typical concentration asdescribed in the table below, and then allowed to incubate at roomtemperature for 20 minutes. At the end of this time, a combination ofquench (EDTA) and detection (peptide tracer and antibody) reagents wasadded using the protocol of Invitrogen P2748 (Carlsbad, Calif.), aProtein Kinase C Fluorescence polarization Assay Kit. After a 30 minuteperiod of incubation, the amount of phosphorylated peptide generated wasmeasured by fluorescence polarization (Ex=485 nm, Em=535 nm) using aTecan Polarian instrument (Switzerland).

TABLE 4 Peptide Enzyme enzyme substrate SEQ ID source concentration PKCRFARKGSLRQKNV Seq ID Upstate 40 ng/mL theta No. 1 Biotechnologies,Temecula, CA, cat. #14-444 PKC RFARKGSLRQKNV Seq ID  Upstate 50 ng/mLepsilon No. 1 Biotechnologies, Temecula, CA, cat. #14-5182. IL-2 ELISA, Human Primary T Cell, Anti-CD3++CD28+ Assays

Human Primary T Cell Isolation and Culture:

Human primary T cells were prepared as follows. Whole blood was obtainedfrom a healthy volunteer, mixed 1:1 with PBS, layered on to FicollHypaque (Amersham Pharmacia Biotech, Piscataway, N.J., Catalog#17-1440-03) in 2:1 blood/PBS:ficoll ratio and centrifuged for 30minutes at 4° C. at 1750 rpm. The cells at the serum:ficoll interfacewere recovered and washed twice with 5 volumes of PBS. These freshlyisolated human peripheral blood mononuclear cells were cultured inYssel's medium containing 40 U/mL IL2 in a flask pre-coated with 1 μg/mLαCD3 and 5 μg/mL αCD28 (Anti-Human CD3, BD Pharmingen Catalog #555336,Anti-Human CD28, Beckman Coulter Catalog #1M1376). The cells werestimulated for 3-4 days, then transferred to a fresh flask andmaintained in RPMI (RPMI-1640 with L-Glutamine; Mediatech, Inc., HerndonVa., cat. #10-040-CM) with 10% FBS and 40 U/mL IL-2. The primary T-cellswere then washed twice with PBS to remove the IL-2.

Primary T Cell Stimulation and IL2 ELISA:

Human primary T cells (100,000 cells per well) were pre-incubated withor without test compound in Yssel's medium for 1 hr at 37° C. Cells werethen stimulated by transferring them to round-bottom 96-well platespre-coated with 1 μg/ml αCD3 and 5 μg/ml αCD28. For counter assay, cellswere instead stimulated by adding 8× stock solutions of PMA andionomycin in Yssels (for final concentrations of 0.5 ng/ml PMA and 0.1μM ionomycin, both from Calbiochem). Cells were incubated at 37° C. for24 hours before 100 μL supernatants were harvested for quantification ofIL-2 by ELISA using Human IL-2 Duoset ELISA Kit from R and D Systems,Cat. #DY202E.

3. Protein Kinase C Assay

The subject compounds can be tested for activity on different PKCisoforms according to the following method. Assay is performed in awhite with clear bottom 384-well microtiterplate with non-bindingsurface. The reaction mixture (25 μl) contains 1.5 μM of atridecapeptide acceptor substrate that mimics the pseudo substratesequence of PKC α with the Ala→Ser replacement, 10 μM ³³P-ATP, 10 mMMg(NO₃)₂, 0.2 mM CaCl₂, PKG at a protein concentration varying from 25to 400 ng/ml (depending on the isotype used), lipid vesicles (containing30 mol % phosphatidylserine, 5 mol % DAG and 65 mol %phosphatidylcholine) at a final lipid concentration of 0.5 mM, in 20 mMTris-HCl buffer pH 7.4+0.1% BSA. Incubation is performed for 60 minutesat room temperature. Reaction is stopped by adding 50 μl of stop mix(100 mM EDTA, 200 μM ATP, 0.1% Triton X-100, 0.375 mg/wellstreptavidin-coated SPA beads in phosphate buffered saline w/o Ca, Mg.After 10 minutes incubation at room temperature, the suspension is spundown for 10 minutes at 300 g. Incorporated radioactivity is measured ina Trilux counter for 1 minute. IC₅₀ measurement is performed on aroutine basis by incubating a serial dilution of inhibitor atconcentrations ranging between 1-1000 μM. IC₅₀ values are calculatedfrom the graph by curve fitting with XL Fit® software.

4. Protein Kinase C α Assay

Human recombinant PKC α is obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.

5. Protein Kinase C β1 Assay

Human recombinant PKC β1 is obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.

6. Protein Kinase C δ Assay

Human recombinant PKC 6 is obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.

7. Protein Kinase C ε Assay

Human recombinant PKC ε is obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.

8. Protein Kinase C η Assay

Human recombinant PKC η is obtained from PanVera and is used under theassay conditions as described under Section A.1 above.

9. Protein Kinase C θ Assay

Human recombinant PKC θ is used under the assay conditions as describedabove.

10. CD28 Costimulation Assay

The assay is performed with Jurkat cells transfected with a humaninterleukin-2 promoter/reporter gene construct as described by Baumann Get al. in Transplant. Proc. 1992; 24:43-8, the β-galactosidase reportergene being replaced by the luciferase gene (de Wet J., et al., Mol.Cell. Biol. 1987, 7(2), 725-737). Cells are stimulated by solidphase-coupled antibodies or phorbol myristate acetate (PMA) and the Ca⁺⁺ionophore ionomycin as follows. For antibody-mediated stimulationMicrolite TM1 microtiter plates (Dynatech) are coated with 3 μg/ml goatanti-mouse IgG Fc antibodies (Jackson) in 55 μl phosphate-bufferedsaline (PBS) per well for three hours at room temperature. Plates areblocked after removing the antibodies by incubation with 2% bovine serumalbumin (BSA) in PBS (300 μl per well) for 2 hours at room temperature.After washing three times with 300 μl PBS per well, 10 ng/ml anti-T cellreceptor antibodies (WT31, Becton & Dickinson) and 300 ng/ml anti-CD28antibodies (15E8) in 50 μl 2% BSA/PBS are added as stimulatingantibodies and incubated overnight at 4° C. Finally the plates arewashed three times with 300 μl PBS per well. Seven three-fold serialdilutions of test compounds in duplicates in assay medium (RPMI 1640/10%fetal calf serum (FCS) containing 50 μM 2-mercaptoethanol, 100 units/mlpenicillin and 100 μg/ml streptomycin) are prepared in separate plates,mixed with transfected Jurkat cells (clone K22 290_H23) and incubatedfor 30 minutes at 37° C. in 5% CO₂ 100 μl of this mixture containing1×10⁵ cells are then transferred to the antibody-coated assay plates. Inparallel 100 μl are incubated with 40 ng/ml PMA and 2 μM ionomycin.After incubation for 5.5 hours at 37° C. in 5% CO₂, the level ofluciferase is determined by bioluminescence measurement. The plates arecentrifuged for 10 minutes at 500 g and the supernatant is removed byflicking. Lysis buffer containing 25 mM Tris-phosphate, pH 7.8, 2 mMDTT, 2 mM 1,2-diaminocyclohexane-N,N,N′,N-tetraacetic acid, 10% (v/v)glycerol and 1% (v/v) Triton X-100 is added (20 μl per well). The platesare incubated at room temperature for 10 minutes under constant shaking.Luciferase activity is assessed with a bioluminescence reader(Labsystem, Helsinki, Finland) after automatic addition of 50 μl perwell luciferase reaction buffer containing 20 mM Tricine, 1.07 mM(MgCO₃)₄Mg(OH)₂×5H₂O, 2.67 mM MgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 270 μMcoenzyme A, 470 μM luciferin (Chemie Brunschwig AG), 530 μM ATP, pH 7.8.Lag time is 0.5 seconds, total measuring time is 1 or 2 seconds. Lowcontrol values are light units from anti-T cell receptor- orPMA-stimulated cells, high controls are from anti-T cellreceptor/anti-CD28- or PMA/ionomycin-stimulated cells without any testsample. Low controls are subtracted from all values. The inhibitionobtained in the presence of a test compound is calculated as percentinhibition of the high control. The concentration of test compoundsresulting in 50% inhibition (IC₅₀) is determined from the dose-responsecurves.

11. Bone Marrow Proliferation (BM) Assay

Bone marrow cells from CBA mice (2.5×104 cells per well in flat bottomtissue culture microtiter plates) are incubated in 100 μl RPMI mediumcontaining 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin (GibcoBRL, Basel, Switzerland), 50 tJM 2-mercaptoethanol (Fluke, Buchs,Switzerland), WEHI-3 conditioned medium (7.5% v/v) and L929 conditionedmedium (3% v/v) as a source of growth factors and serially dilutedcompounds. Seven three-fold dilution steps in duplicates per testcompound are performed. After four days of incubation 1 μCi ³H-thymidineis added. Cells are harvested after an additional five-hour incubationperiod, and incorporated ³H-thymidine is determined according tostandard procedures. Conditioned media are prepared as follows. WEHI-3cells 1 (ATCC TIB68) and L929 cells (ATCC CCL 1) are grown in RPMImedium until confluence for 4 days and one week, respectively. Cells areharvested, resuspended in the same culture flasks in medium C containing1% FCS (Schreier and Tees 1981) for WEHI-3 cells and RPMI medium forL929 cells and incubated for 2 days (WEHI-3) or one week (L929). Thesupernatant is collected, filtered through 0.2 μm and stored in aliquotsat −80° C. Cultures without test compounds and without WEHI-3 and L929supernatants are used as low control values. Low control values aresubtracted from all values. High controls without any sample are takenas 100% proliferation. Percent inhibition by the samples is calculatedand the concentrations required for 50% inhibition (IC₅₀ values) aredetermined.

12. Allogeneic Mixed Lymphocyte Reaction (MLR)

The two-way MLR is performed according to standard procedures (J.Immunol. Methods, 1973, 2, 279 and Meo T. et al., Immunological Methods,New York, Academic Press, 1979, 227-39). Briefly, spleen cells from CBAand BALB/c mice (1.6×10⁵ cells from each strain per well in flat bottomtissue culture microtiter plates, 3.2×10⁵ in total) are incubated inRPMI medium containing 10% FCS, 100 U/ml penicillin, 100 μg/mlstreptomycin (Gibco BRL, Basel, Switzerland), 50 μM 2-mercaptoethanol(Fluka, Buchs, Switzerland) and serially diluted compounds. Seventhree-fold dilution steps in duplicates per test compound are performed.After four days of incubation 1 μCi ³H-thymidine is added. Cells areharvested after an additional five-hour incubation period, andincorporated ³H-thymidine is determined according to standardprocedures. Background values (low control) of the MLR are theproliferation of BALB/c cells alone. Low controls are subtracted fromall values. High controls without any sample are taken as 100%proliferation. Percent inhibition by the samples is calculated, and theconcentrations required for 50% inhibition (IC₅₀ values) are determined.

B. In vivo

Heart Transplantation Model

The strain combination used: Male Lewis (RT¹ haplotype) and BN(RT¹haplotype). The animals are anaesthetised using inhalationalisofluorane. Following heparinisation of the donor rat through theabdominal inferior vena cava with simultaneous exsanguination via theaorta, the chest is opened and the heart rapidly cooled. The aorta isligated and divided distal to the first branch and the brachiocephalictrunk is divided at the first bifurcation. The left pulmonary artery isligated and divided and the right side divided but left open. All othervessels are dissected free, ligated and divided and the donor heart isremoved into iced saline.

The recipient is prepared by dissection and cross-clamping of theinfra-renal abdominal aorta and vena cava. The graft is implanted withend-to-side anastomoses, using 1010 monofilament suture, between thedonor brachiocephalic trunk and the recipient aorta and the donor rightpulmonary artery to the recipient vena cava. The clamps are removed, thegraft tethered retroabdominally, the abdominal contents washed with warmsaline and the animal is closed and allowed to recover under a heatinglamp. Graft survival is monitored by daily palpation of the beatingdonor heart through the abdominal wall. Rejection is considered to becomplete when-heart beat stops. Graft survival is monitored in animalstreated with compounds.

Graft v. Host Model

Spleen cells (2×10⁷) from Wistar/F rats are injected subcutaneously intothe right hind footpad of (Wistar/F×Fischer 344) F₁ hybrid rats. Theleft footpad is left untreated. The animals are treated with the testcompounds on 4 consecutive days (0-3). The popliteal lymph nodes areremoved on day 7, and the weight differences between two correspondinglymph nodes are determined. The results are expressed as the inhibitionof lymph node enlargement (given in percent) comparing the lymph nodeweight differences in the experimental groups to the weight differencebetween the corresponding lymph nodes from a group of animals leftuntreated with a test compound. In certain instances the test compoundis a selective PKC inhibitor. For example, disclosed compounds that areparticularly useful for treating graft versus host disease and relateddisorders are selective PKC α and θ inhibitors.

Research Applications

Since subject compounds can inhibit a PKC activity, such compounds arealso useful as research tools. The present disclosure also provides amethod for using a compound of formula (I) or a salt or solvate orstereoisomer thereof as a research tool for studying a biological systemor sample, or for discovering new chemical compounds that can inhibit aPKC activity.

The disclosure provides for a method of studying a biological system orsample known to comprise PKC, the method comprising: (a) contacting thebiological sample with a compound of formula (I) or a salt or solvate orstereoisomer thereof; and (b) determining the inhibiting effects causedby the compound on the biological sample.

Any suitable biological sample having PKC can be employed in suchstudies which can be conducted either in vitro or in vivo.Representative biological samples suitable for such studies include, butare not limited to, cells, cellular extracts, plasma membranes, tissuesamples, isolated organs, mammals (such as mice, rats, guinea pigs,rabbits, dogs, pigs, humans, and so forth), and the like, with mammalsbeing of particular interest.

When used as a research tool, a biological sample comprising PKC istypically contacted with a PKC activity-inhibiting amount of a subjectcompound. After the biological sample is exposed to the compound, theeffects of inhibition of a PKC activity are determined usingconventional procedures and equipment, such as the assays disclosedherein. Exposure encompasses contacting the biological sample with thecompound or administering the compound to a subject. The determiningstep can involve measuring a response (a quantitative analysis) or caninvolve making an observation (a qualitative analysis). Measuring aresponse involves, for example, determining the effects of the compoundon the biological sample using conventional procedures and equipment,such as radioligand binding assays and measuring ligand-mediated changesin functional assays. The assay results can be used to determine theactivity level as well as the amount of compound necessary to achievethe desired result, that is, a PKC activity-inhibiting amount.

Additionally, subject compounds can be used as research tools forevaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds having a PKCinhibiting activity. In this manner, a subject compound can be used as astandard in an assay to allow comparison of the results obtained with atest compound and with the subject compounds to identify those testcompounds that have about equal or superior activity, if any. Forexample, IC₅₀ data for a test compound or a group of test compounds iscompared to the IC₅₀ data for a subject compound to identify those testcompounds that have the desired properties, for example, test compoundshaving an IC₅₀ about equal or superior to a subject compound, if any.

This aspect includes, as separate embodiments, both the generation ofcomparison data (using the appropriate assays) and the analysis of testdata to identify test compounds of interest. Thus, a test compound canbe evaluated in a biological assay, by a method comprising the steps of:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a subjectcompound to provide a second assay value; wherein step (a) is conductedeither before, after or concurrently with step (b); and (c) comparingthe first assay value from step (a) with the second assay value fromstep (b). The assays that can be used for generation of comparison dataare disclosed herein, such as the PKC assays.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Celsius, and pressure is ator near atmospheric. Standard abbreviations may be used.

Examples 1 and 2

Preparation 1 Synthesis of 1-(2-fluoro-5-nitrophenyl)-1H-pyrrole (P-1)

5-Nitro-2-fluoroaniline (7.0 g, 44.8 mmol) was taken up in acetic acid(60 mL) in a 250 mL round bottomed flask and treated with2,5-dimethoxytetrahydrofuran (6.3 mL, 49.8 mmol). The resulting mixturewas heated to 110° C. for 4 hours. TLC analysis indicated completeconversion of starting material to product after 4 hours. Thus, thereaction mixture was cooled to ambient temperature, transferred to aseparatory funnel and partitioned (CH₂Cl₂//H₂O). The organic phase waswashed with H₂O (3×50 mL), brine (3×50 mL), dried over Na₂SO₄ andevaporated in vacuo to give a light brown residue. The residue waspurified by flash chromatography (hexanes→1:9 EtOAc/hexanes) to affordthe title compound as a light yellow solid (7.2 g, 78%).

¹H NMR (DMSO-d₆, 300 MHz) 8.34 (dd, 1H), 8.16-8.20 (m, 1H), 7.69 (t,1H), 7.27 (d, 2H), 6.32 (d, 2H) ppm; MS (ES) 207 (M+H)

Preparation 2 Synthesis of1-(2-fluoro-5-nitrophenyl)-1H-pyrrole-2-carbaldehyde (P-2)

Anhydrous DMF (0.82 mL, 10.67 mmol) and POCl₃ (0.96 mL, 10.67 mmol) werecombined at 0° C. in a 50 mL round bottomed flask. The suspension waswarmed to ambient temperature and treated with1-(2-fluoro-5-nitrophenyl)-1H-pyrrole (2.0 g, 9.7 mmol) as a lightyellow solution in anhydrous DMF (8 mL). The resulting mixture wasvigorously stirred at room temperature for 5 hours. TLC analysis of themixture after 5 hours showed the complete conversion of the startingmaterial to the product. The reaction mixture was transferred to aseparatory funnel and partitioned (EtOAc//H₂O). The organic phase waswashed with H₂O (3×50 mL), brine (3×50 mL), dried over Na₂SO₄ andevaporated in vacuo to give a brown residue. The residue was purified byflash chromatography (hexanes 1:1 EtOAc/hexanes) to furnish the titlecompound as a white solid (1.2 g, 54%).

¹H NMR (DMSO-d₆, 300 MHz) 9.53 (s, 1H), 8.40 (dd, 1H), 7.70 (t, 1H),7.51 (s, 1H), 7.32 (d, 1H), 6.54 (t, 1H); MS (ES) 235 (M+H).

Preparation 3 Synthesis of 8-nitro-4H-benzo[b]pyrrolo[1,2-d][1,4]oxazine(P-3)

1-(2-fluoro-5-nitrophenyl)-1H-pyrrole-2-carbaldehyde (3.95 g, 16.9 mmol)was suspended in ethanol (100 mL) in a 250 mL round bottomed flask andtreated with sodium borohydride (767 mg, 20.3 mmol, 1.2 equiv.). Theresulting mixture was stirred at 60° C. for 5 hours. The reactionmixture was cooled to ambient temperature, transferred to a separatoryfunnel and partitioned (CH₂Cl₂//H₂O). The organic phase was washed withH₂O (3×50 mL), brine (3×50 mL), dried over Na₂SO₄ and evaporated invacuo to give a light yellow residue. The residue was purified by flashchromatography (hexanes 1:9 EtOAc/hexanes) to afford the title compoundas a light yellow solid (2.5 g, 68%).

¹H NMR (DMSO-d₆, 300 MHz) 8.53 (d, 1H), 7.96 (dd, 1H), 7.77 (s, 1H),7.26 (d, 1H), 6.35 (t, 1H), 6.14 (s, 1H), 5.33 (s, 2H); MS (ES) 217(M+H).

Preparation 4 Synthesis of 4H-benzo[b]pyrrolo[1,2-d][1,4]oxazin-8-amine(P-4)

¹H NMR (DMSO-d₆, 300 MHz) 7.21 (t, 1H), 6.76 (s, 1H), 6.74 (d, 1H), 6.29(dd, 1H), 6.23 (t, 1H), 6.01 (s, 1H), 4.97 (s, 2H), 4.88 (s, 2H); MS(ES) 187 (M+H).

Example 1 Synthesis ofN2-(4H-benzo[b]pyrrolo[1,2-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine(Compound 1-4)

¹H NMR (DMSO-d₆, 300 MHz) 8.88 (s, 1H), 8.34 (s, 1H), 7.85 (d, 1H), 7.46(d, 1H), 7.18-7.19 (m, 2H), 6.86 (d, 1H), 6.23-6.28 (m, 2H), 6.05 (s,1H), 5.05 (s, 2H), 4.35-4.40 (m, 1H), 1.78 (d, 2H), 1.35 (t, 2H), 1.20(s, 6H), 1.17 (s, 6H); MS (ES) 437 (M+H).

Example 2 Spectral data forN2-(4H-benzo[b]pyrrolo[1,2-d][1,4]oxazin-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine(Compound 1-3)

¹H NMR (DMSO-d₆, 300 MHz) 8.83 (s, 1H), 7.83 (d, 1H), 7.66 (d, 1H), 7.52(dd, 1H), 7.23 (s, 1H), 7.14 (d, 1H), 6.86 (d, 1H), 6.25 (t, 1H), 6.05(s, 1H), 5.06 (s, 2H), 4.31-4.33 (m, 1H), 2.15 (s, 3H), 1.67 (dd, 2H),1.43 (t, 2H), 1.05 (s, 6H), 0.97 (s, 6H); MS (ES) 451 (M+H).

Examples 3-6

Example 3 Synthesis ofN2-(4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-((1,2,2,5,5-pentamethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine

¹H NMR (DMSO-d6, 300 MHz) 9.36 (s, 1H), 8.59 (s, 1H), 7.85 (s, 1H), 7.59(d, 1H), 7.48 (s, br, 1H), 7.15 (d, 1H), 5.77 (s, 2H), 2.19 (m, 1H),2.15 (s, 3H), 1.85 (m, 1H), 1.45 (t, 1H), 1.20 (dd, 2H), 1.05 (s, 6H),0.92 (s, 3H), 0.80 (s, 3H) ppm; MS (ES) 454.52 (M+H).

Example 4 Synthesis ofN2-(4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-((2,2,5,5-tetramethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine

¹H NMR (DMSO-d6, 300 MHz) 9.39 (s, 1H), 8.60 (s, 1H), 7.88 (s, 1H), 7.55(d, 1H), 7.45 (s, br, 1H), 7.19 (d, 1H), 5.75 (s, 2H), 3.48 (m, br, 1H),2.10 (m, 1H), 1.90 (m, 1H), 1.65 (m, 1H), 1.30 (dd, 2H), 1.20 (s, 6H),1.15 (s, 3H), 1.10 (s, 3H) ppm; MS (ES) 440.49 (M+H).

Example 5 Synthesis ofN2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-((1,2,2,5,5-pentamethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine

¹H NMR (DMSO-d6, 300 MHz) 9.32 (s, 1H), 8.60 (s, 1H), 7.88 (s, 1H), 7.58(d, 1H), 7.44 (s, br, 1H), 7.10 (d, 1H), 3.50 (m, 1H), 2.10 (m, 1H),2.05 (s, 3H), 1.80 (m, 1H), 1.72 (s, 6H), 1.42 (t, 1H), 1.04 (m, 1H),1.00 (s, 6H), 0.92 (s, 3H), 0.80 (s, 3H) ppm; MS (ES) 482.57 (M+H).

Example 6 Synthesis ofN2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-((2,2,5,5-tetramethylpyrrolidin-3-yl)methyl)pyrimidine-2,4-diamine

¹H NMR (DMSO-d6, 300 MHz) 9.34 (s, 1H), 8.62 (s, 1H), 7.88 (s, 1H), 7.56(d, 1H), 7.46 (s, br, 1H), 7.12 (d, 1H), 3.45 (m, 1H), 2.24 (m, 1H),1.80 (m, 1H), 1.70-1.75 (m, 7H), 1.56 (t, 2H), 1.09 (s, 3H), 1.06 (s,3H), 1.02 (s, 3H), 0.95 (s, 3H) ppm; MS (ES) 468.54 (M+H).

Examples 7-8

Compounds 1-1 and 1-2 were synthesized according to the followingscheme:

Example 7 Synthesis ofN2-(4H-benzo[13]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine(Compound 1-1)

¹H NMR (DMSO-d₆, 300 MHz) 8.65 (d, 1H), 8.26 (d, 1H), 8.01 (d, 1H), 7.81(bs, 1H), 7.76 (bs, 1H), 7.63 (dd, 1H), 7.19 (d, 1H), 5.72 (s, 2H), 4.47(m, 1H), 2.05-1.85 (d, 2H), 1.65-1.51 (t, 2H), 1.42 (d, 12H) ppm; MS(ES) 440 (M+H)9

Example 8 Synthesis ofN2-(4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine(Compound 1-2)

¹H NMR (DMSO-d₆, 300 MHz) 9.51 (s, 1H), 8.32 (s, 1H), 8.01 (d, 1H), 7.91(d, 1H), 7.36 (d, 1H), 7.09 (d, 1H), 5.72 (s, 2H), 4.47 (m, 1H), 2.28(s, 3H), 2.11-2.05 (d, 2H), 1.85-1.71 (t, 2H), 1.35 (d, 12H) ppm; MS(ES) 454 (M+H).

Compounds 1-7 and 1-8 were prepared in similar fashion to Compounds 1-1and 1-2 as illustrated above.

Examples 9 and 10

This Example describes the synthesis of Compounds 1-11 and 1-12. Thesynthesis is analogous to that set forth in Examples 7 and 8. Thefollowing scheme outlines the synthesis used for these compounds:

Preparation 5 Synthesis of2,1-Spiro-butane-6-nitro-2H-benzo[b][1,4]oxazine-3(4H)-one (P-5)

¹H NMR (DMSO, 300 MHz) 11.0 (s, 1H), 8.4-8.1 (d, J=8.7 Hz, 1H), 7.7 (s,1H), 7.21-7.18 (d, J=8.7 Hz, 1H), 2.54 (bs (2H), 2.34-2.24 (m, 2H),1.96-1.89 (m, 1H), 1.84-1.75 (m, 1H)ppm; MS (ES) 235.23 (M+H)

Preparation 6 Synthesis of2,1-Spiro-butane-6-nitro-2H-benzo[b][1,4]oxazine-3(4H)-thione (P-6)

¹H NMR (DMSO, 300 MHz) 7.95-7.93 (d, J=8.7 Hz, 1H), 7.89 (s, 1H),7.29-7.26 (d, J=9.0 Hz, 1H), 2.78-2.71 (m, 2H), 2.4-2.3 (m, 2H),1.99-1.85 (m, 2H)ppm; MS (ES) 251.23 (M+H)

Preparation 7 Synthesis of2,1-Spiro-butane-8-nitro-4H-tetrazolo[5,1-c][1,4]benzoxazine (P-7)

¹H NMR (DMSO, 300 MHz) 8.57 (s, 1H), 8.29-8.26 (d, J=9.3 Hz, 1H),7.5-7.47 (d, J=9.0 Hz, 1H), 2.78-2.73 (t, J=8.1 Hz, 4H), 2.1-2.05 (m,2H)ppm; MS (ES) 260.27 (M+H)

Preparation 8 Synthesis of8-Amino-2,1-spiro-butane-4H-tetrazolo[5,1-c][1,4]benzoxazine (P-8)

¹H NMR (DMSO, 300 MHz) 7.09 (s, 1H), 7.0-6.97 (d, J=8.7 Hz, 1H),6.59-6.56 (d, J=9.0 Hz, 1H), 5.36 (s, 2H), 2.61-2.55 (m, 4H), 2.02-1.96(m, 2H)ppm; MS (ES) 230.24 (M+H)

Example 9 Synthesis ofN2-{2,1-Spiro-butane-4H-tetrazolo[5,1-c][1,4]benzoxazin-8-yl}-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pyrimidinediamine(Compound 1-12)

¹H NMR (DMSO, 300 MHz) 9.25 (s, 1H), 8.24 (s, 1H), 7.88-7.87 (d, J=3.6Hz, 1H), 7.77-7.74 (d, J=9.0 Hz, 1H), 7.23-7.2 (d, J=8.4 Hz, 1H),7.13-7.1 (d, J=8.7 Hz, 1H), 4.36 (bs, 1H), 2.66-2.61 (t, J=8.1 Hz, 4H),2.13 (s, 3H), 2.05-2.02 (m, 2H), 1.7-1.67 (d, J=10.2 Hz, 2H), 1.47-1.39(t, J=12.3 Hz, 2H), 1.04 (s, 6H), 0.97 (s, 6H)ppm; MS (ES) 495.58 (M+H)

Example 10 Synthesis ofN2-{2,1-Spiro-butane-4H-tetrazolo[5,1-c][1,4]benzoxazin-8-yl}-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)-2,4-pyrimidinediamine(Compound 1-13)

¹H NMR (DMSO, 300 MHz) 9.25 (s, 1H), 8.24 (s, 1H), 7.88-7.86 (d, J=3.9Hz, 1H), 7.76-7.73 (d, J=9.0 Hz, 1H), 7.23-7.2 (d, J=8.7 Hz, 1H),7.13-7.1 (d, J=9.0 Hz, 1H), 4.38 (bs, 1H), 2.65-2.6 (t, J=7.5, 4H),2.03-2.01 (m, 2H), 1.71-1.68 (d, J=9.9 Hz, 2H), 1.19-1.14 (t, J=7.5 Hz,2H), 1.1 (s, 6H), 1.01 (s, 6H)ppm; MS (ES) 480.50 (M+H)

Examples 11-12

Preparation 9 Synthesis of2,2-difluoro-6-nitro-2H-benzo[b][1,4]oxazine-3(4H)-thione (P-9)

¹H NMR (DMSO, 300 MHz) 8.1-8.07 (d, J=9.0 Hz, 1H), 8.05-8.04 (d, J=2.7Hz, 1H), 7.63-7.6 (d, J=9.0 Hz, 1H)ppm; MS (ES) 247.18 (M+H)

Preparation 10 Synthesis of4,4-difluoro-8-nitro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazine (P-10)

¹H NMR (DMSO, 300 MHz) 8.82 (s, 1H), 8.49-8.46 (d, J=9.3 Hz, 1H),7.95-7.92 (d, J=9.0 Hz, 1H)ppm; MS (ES) 256.12 (M+H)

Preparation 11 Synthesis of4,4-difluoro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-amine (P-11)

¹H NMR (DMSO, 300 MHz) 7.3-7.27 (d, J=9.0 Hz, 1H), 7.26 (s, 1H),6.75-6.72 (d, J=9.0 Hz, 1H), 5.7 (s, 2H)ppm; MS (ES) 226.19 (M+H)

Example 11 Synthesis ofN2-(4,4-difluoro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine

¹H NMR (DMSO, 300 MHz) 9.45 (s, 1H), 8.5 (s, 1H), 7.98-7.92 (m, 2H),7.47-7.44 (d, J=9.0 Hz, 1H), 7.31-7.29 (d, J=7.5 Hz, 1H), 4.36 (bs, 1H),2.18 (s, 3H), 1.73-1.69 (d, J=11.7 Hz, 2H), 1.51-1.43 (t, J=11.7 Hz,2H), 1.07 (s, 6H), 1.03 (s, 6H)ppm; MS (ES) 490.07 (M+H)

Example 12 Synthesis ofN2-(4,4-difluoro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine

¹H NMR (DMSO, 300 MHz) 9.47 (s, 1H), 8.52 (s, 1H), 7.95-7.92 (m, 2H),7.49-7.46 (d, J=9.0 Hz, 1H), 7.42-7.39 (d, J=8.4 Hz, 1H), 4.46 (bs, 1H),1.82-1.76 (d, J=9.9 Hz, 2H), 1.38-1.3 (t, J=12.3 Hz, 2H), 1.26 (s, 6H),1.16 (s, 6H)ppm; MS (ES) 476.10 (M+H)

Examples 13-14

Preparation 12 Synthesis of2,2-dimethyl-6-nitro-2H-benzo[b][1,4]oxazine-3(4H)-thione (P-12)

¹H NMR (DMSO, 300 MHz) 7.95-7.92 (m, 2H), 7.19-7.17 (d, J=8.1 Hz, 1H),1.59 (s, 6H)ppm; MS (ES) 239.20 (M+H)

Preparation 13 Synthesis of4,4-dimethyl-8-nitro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazine (P-13)

¹H NMR (DMSO, 300 MHz) 8.6 (s, 1H), 8.3-8.27 (d, J=9.3 Hz, 1H),7.48-7.45 (d, J=9.0 Hz, 1H), 1.83 (s, 6H)ppm; MS (ES) 248.22 (M+H)

Preparation 14 Synthesis of4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-amine (P-14)

¹H NMR (DMSO, 300 MHz) 7.1 (s, 1H), 6.94-6.91 (d, J=8.7 Hz, 1H),6.59-6.57 (d, J=8.7 Hz, 1H), 5.34 (s, 2H), 1.67 (s, 6H)ppm; MS (ES)218.28 (M+H)

Example 13 Synthesis ofN2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine

¹H NMR (DMSO, 300 MHz) 9.25 (s, 1H), 8.27 (s, 1H), 7.88-7.86 (d, J=3.9Hz, 1H), 7.74-7.71 (d, J=9.0 Hz, 1H), 7.21-7.19 (d, J=7.8 Hz, 1H),7.08-7.05 (d, J=9.0 Hz, 1H), 4.35 (bs, 1H), 2.12 (s, 3H), 1.72-1.67 (m,8H), 1.47-1.39 (t, J=12.3 Hz, 2H), 1.04 (s, 6H), 0.95 (s, 6H)ppm; MS(ES) 482.34 (M+H)

Example 14 Synthesis ofN2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(2,2,6,6-tetramethylpiperidin-4-yl)pyrimidine-2,4-diamine

¹H NMR (DMSO, 300 MHz) 9.25 (s, 1H), 8.27 (s, 1H), 7.88-7.87 (d, J=3.6Hz, 1H), 7.74-7.7 (d, J=9.3 Hz, 1H), 7.25-7.22 (d, J=8.1 Hz, 1H),7.08-7.05 (d, J=9.0 Hz, 1H), 4.39 (bs, 1H), 1.71 (bs, 8H), 1.23-1.14 (t,J=12.6 Hz, 2H), 1.11 (s, 6H), 1.04 (s, 6H)ppm; MS (ES) 468.10 (M+H)

Example 15 Assays of Exemplary Compounds

Several subject compounds were tested according to procedures in the“Characterization of Functional Properties” section. PKC kinase activitywas measured using the procedure Section A.1 (Protein Kinase C assay)with the appropriate isoform. The whole cell assay was run according tothe procedure in Section A.2 (IL-2 ELISA, Human primary T cell,anti-CD3+CD28+ Assays). Assay data for certain compounds from Tables 1and 2 are listed below in Table 5, in which “A” indicates an IC₅₀ in theindicated assay of less than 0.25 μM; “B” is 0.25 to 0.5 μM; “C” is 0.5to 1 μM; and “D” is from 1 μM to 5 μM. Blank entries indicate that theIC₅₀ was not determined.

TABLE 5 Whole PKC- PKC- PKC- PKC- PKC- Compound cell assay alpha betadelta epsilon theta 1-1 A A A A A A 1-2 A A A A A A 1-3 D 1-4 D 1-5 C AA A A A 1-6 B A A A A A 1-7 A A A A A A 1-8 A A A A A A 1-9 C  1-10 A 1-11 A A A A A A  1-12 A A A A A A  1-17 A A A A A A  1-18 A A A A A A2-1 A A A A A A 2-2 A A A A A A 2-7 A A A A A A 2-8 B A B A A A  2-13 AA A A A A  2-14 A A A A A A

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for treating a lymphoid neoplasm,comprising administering to a subject in need thereof an effectiveamount ofN2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(1,2,2,6,6-pentamethylpiperidin-4-yl)pyrimidine-2,4-diamine.2. The method of claim 1, wherein the lymphoid neoplasm is a B-cellneoplasm, a T-cell or NK-cell neoplasm, a Hodgkin's lymphoma, or acombination thereof.
 3. The method of claim 1, wherein the lymphoidneoplasm is a B-cell neoplasm.
 4. The method of claim 3, wherein theB-cell neoplasm is selected from the group consisting of a precursorB-lymphoblastic leukemia/lymphoma, a B-cell chronic lymphocyticleukemia/small lymphocytic lymphoma, a B-cell prolymphocytic leukemia, alymphoplasmacytic lymphoma, a splenic marginal zone B-cell lymphoma, ahairy cell leukemia, a plasma cell myeloma/plasmacytoma, an extranodalmarginal zone B-cell lymphoma of MALT type, a nodal marginal zone B-celllymphoma, a follicular lymphoma, a mantle-cell lymphoma, a diffuse largeB-cell lymphoma, a mediastinal large B-cell lymphoma, a primary effusionlymphoma, and a Burkitt's lymphoma/Burkitt cell leukemia.
 5. The methodof claim 1, wherein the lymphoid neoplasm is a T-cell or NK-cellneoplasm.
 6. The method of claim 5, wherein the T-cell or NK-cellneoplasm is selected from the group consisting of a precursorT-lymphoblastic lymphoma/leukemia, a T-cell prolymphocytic leukemiaT-cell granular lymphocytic leukemia, an aggressive NK-cell leukemia, anadult T-cell lymphoma/leukemia (HTLV-1), an extranodal NK/T-celllymphoma, a nasal type T-cell lymphoma, an enteropathy-type T-celllymphoma, a hepatosplenic gamma-delta T-cell lymphoma, a subcutaneouspanniculitis-like T-cell lymphoma, a Mycosis fungoides/Sezary syndrome,an Anaplastic large-cell lymphoma, a T/null cell, a primary cutaneoustype, a Peripheral T-cell lymphoma, an Angioimmunoblastic T-celllymphoma, an Anaplastic large-cell lymphoma, and a primary systemic typeT-cell lymphoma.
 7. The method of claim 1, wherein the lymphoid neoplasmis a Hodgkin's lymphoma.
 8. The method of claim 7, wherein the Hodgkin'slymphoma is selected from the group consisting of a nodularlymphocyte-predominant Hodgkin's lymphoma, a Nodular sclerosis Hodgkin'slymphoma (grades 1 and 2), a Lymphocyte-rich classical Hodgkin'slymphoma, a mixed cellularity Hodgkin's lymphoma, and a lymphocytedepletion Hodgkin's lymphoma.